Ecology

The haplotype networks, PCoA analysis and STRUCTURE analysis based on the six nuclear loci confirm that S. longifolium is a hybrid between S. emersum and S. gramineum, providing molecular support for previous morphological analyses5. Furthermore, all individuals with intermediate admixture coefficient (Fig. 2b) and private haplotypes only present in one out of six nuclear loci (Fig. 1) suggest that S. longifolium is most likely a F1 hybrid. We thus hypothesized that S. emersum and S. gramineum could likely maintain their species boundary through the post-zygote reproductive isolation mechanism of F1 generation sterility. This hypothesis is possible based on the observations from hybrids in European Russia. The pollen viability was checked in S. longifolium samples from Vysokovskoe Lake and Sabro Lake, and the vast majority of checked pollens were sterile5. In addition, flowering plants of S. longifolium often do not form seeds, or the seeds are puny and significantly inferior to normal seeds in size5. However, the hypothesis is only based on our limited sampling, which is contrary to the conclusion inferred from morphological characteristics that it is fertile and may backcross with parental species1. Further studies with extensive sampling are necessary to test our hypothesis.The chloroplast DNA fragment trnH-psbA was used to infer the direction of hybridization between S. emersum and S. gramineum because chloroplast DNA is maternal inheritance in Sparganium3,4. The hybrid S. longifolium shared haplotypes with S. emersum and S. gramineum simultaneously (Fig. 1). This finding clearly indicates that bidirectional hybridization exists between S. emersum and S. gramineum. At the same time, the different frequency of these two haplotypes in the hybrid (H1, 19.1% vs. H2, 80.9%) means that the direction of hybridization is asymmetric. A variety of factors can lead to asymmetry in natural hybridization, such as flowering time, preference of pollinators, quality and quantity of pollen, cross incompatibility and the abundance of parent species7,8. Rare species usually act as maternal species relative to abundant species9,10. S. gramineum is confined to oligotrophic lakes and its abundance is obviously lower than that of S. emersum1,11. The relatively scarcity combined with the ecology of S. gramineum make it more often act as maternal species when hybridizing with S. emersum.As described by5, the morphological diversification of S. longifolium was also observed in this study. For example, individuals of S. longifolium with emergent and floating-leaved life forms occur concurrently in Zaozer’ye Lake (Supplementary Fig. S2). However, all individuals had the same haplotype H2 as S. gramineum (Fig. 1), suggesting that the direction of hybridization do not determine life form of S. longifolium. In addition, all individuals of S. longifolium sampled here are likely F1 hybrid. Their variable phenotypes could not be associated with traits segregation due to F2 generation or backcross. Detailed ecological investigation combining with research at the genomic level are essential to find out the potential factors leading to morphological diversification of S. longifolium.Here, using sequences of six nuclear loci and one chloroplast DNA fragment, we confirmed that S. longifolium is the hybrid between S. emersum and S. gramineum. The natural hybridization between S. emersum and S. gramineum is bidirectional but the latter mainly acts as maternal species. We also found that all samples of S. longifolium were F1 generations, indicating that S. emersum and S. gramineum could maintain their species boundary through the post-zygote reproductive isolation mechanism of F1 generation sterility. More

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Influence of the PLHP on water depth distributionThe hydrodynamic process of Poyang Lake with and without the PLHP is simulated by M1 and M2, respectively. By comparing and analyzing the simulation results, we obtain the changes of the water depth, i.e., the water depth in M2 minus the water depth in M1, in Poyang Lake. Figure 5 shows the mean monthly water depth differences during September and October in three typical years. The water depth in Poyang Lake has increased obviously in most cases after the operation of the project. Combining the water level processes in Fig. 4 and the water depth differences in Fig. 5, the area of the changes in the water depth is seen to be mainly controlled by the Higher Water Levels (HWL) between M1 and M2. While the magnitude of the changes is mainly controlled by the Differences in Water Levels (DWL) between M1 and M2.Figure 5Water depth variation during September and October in the typical years. The figure was generated by Tecplot2020 (https://www.tecplot.com/).Full size imageAs shown in the water level variations processes in Fig. 4a, in the low-water-level year (2006), where the natural inflow is relatively small, the water level in M2 is much higher than that in M1 and reaches the peak value around 10th of October. The DWL also reaches maximum in this time and then gradually decreases. As a consequence, the water depth increases the most in October 2006. As shown in Fig. 5d, in most areas of Poyang Lake, the Xiuhe River and the Ganjiang River, the water depth is increased by more than 1 m. Especially in the main channel from the PLHP to Tangyin and Wucheng, the increase can exceed 4 m, as shown in the red parts in Fig. 5d. While over the surrounding flooded land, the increase ranges from 1 to 3 m. The increase in the water depth during September was essentially the same as that in October, but the area and magnitude of the changes are slightly reduced. The maximum increase is about 3.1 m, which is mainly concentrated in the main channel on the north of Songmen Mountain.In the medium-water-level year (2018), the water level in M1 is constantly lower than that in M2 during September and October, which can be seen in Fig. 4b. Both the HWL and DWL first increase and then decrease, reaching their maximum values in early September and late September, respectively. Furthermore, the mean monthly HWL and DWL in September are slightly greater than those in October. As a consequence, the area and magnitude of the changes in Fig. 5b are larger than those in Fig. 5e. In September 2018, almost the entire main lake region becomes influenced by the PLHP and the increase in water depth ranges from 0 to 3.4 m. While in October 2018, the increase in water depth ranges from 0 to 3.2 m, and the significant increase is mainly concentrated in the main channel on the north of Songmen Mountain.In the high-water-level year (2010), as shown in Fig. 4c, the HWL rises and the DWL decreases as compared to those in other years. During the first 35 days, the hydrodynamic conditions in M2 are exactly the same as that in M1, so the PLHP has no effect on the water depth in the lake region in September (Fig. 5c). However, after the 6th of October, the water level in M2 begins to be higher than that in M1 under the regulation of the PLHP. Although the DWL is small during this time, the HWL is relatively high and thus results in large areas of water depth increase, as shown in Fig. 5f. The maximum increase in water depth is approximately 1.1 m.In these two months, the northeastern parts of the two national nature reserves are observably influenced., The area of the changes is greatest in September 2018, there has been a marked increase in water depth in most areas of the reserves, with the maximum increase reaching about 2.8 m. While in other periods during September and October, with the exception of September 2010, the water depth is increased significantly in about 1/3 ~ 1/2 area of the reserves, with the maximum increase reaching about 2.6 m. In the meantime, the water depth in the southwestern part of these two reserves remains essentially the same as before the operation of the PLHP, with an increase of less than 0.25 m as shown in the grey parts in Fig. 5.Influence of the PLHP on habitat suitability of Vallisneria natansAccording to the relationship between the habitat suitability of Vallisneria natans and the water depth as mentioned in Fig. 3, the water depth results can be translated into the habitat suitability of Vallisneria natans, as shown in Fig. 6. The first and third rows are the distributions of the habitat suitability during September and October, respectively, in the three typical years before the operation of the PLHP, while the second and fourth rows are distributions of the habitat suitability after the operation of the PLHP. The grey parts in Fig. 6 indicate that the habitat suitability is 0, implying that the area is dry or the water depth is greater than 4 m.Figure 6Distributions of the habitat suitability of Vallisneria natans in typical years without (the first and third rows) and with (the second and fourth rows) the PLHP. The figure was generated by Tecplot2020 (https://www.tecplot.com/).Full size imageAs shown in Fig. 6, the suitable area for the growth of Vallisneria natans is mainly concentrated over the flooded land. As the water depth in the main channel is generally more than 4 m, so the habitat suitability is usually 0 there.Before the operation of the PLHP (M1), the water level in 2010 is higher than that those in 2006 and 2018. Therefore, there are more areas covered by water in 2010, and the habitat suitability in Fig. 6c is greater than those in Fig. 6a,b. Similarly, the habitat suitability in Fig. 6i is greater than those in Fig. 6g,h. Because the lake bed is lower in northeastern part than that in the southwestern part, the water depth generally decreases from northeast to southwest. During September and October in 2006 and 2018, large areas of bed in the northeastern part are covered by water, and the water depth is less than 4 m, which is suitable for the growth of Vallisneria natans. While large areas in the southwestern part of the lake are dry, and thus the habitat suitability is 0, as shown in the grey parts in the southwestern part of the main lake region in Fig. 6a,b,g,h. However, because the water level is relatively high during September and October in 2010, there are almost no dry areas in the lake region. As a result, with the exception of the main channel, where the water depth is greater than 4 m, most of the areas in the lake region are suitable for the growth of Vallisneria natans. The most suitable areas for Vallisneria natans vary between these two months, as the water depth in September 2010 is greater than that in October 2010. As shown in Fig. 6c, the red parts are mainly concentrated in the southwestern part of the lake, because there are large areas of flooded region with water depths ranging from 1 to 2 m, which is ideal for the growth of Vallisneria natans. On the contrary, the water depth in the northeastern flood land is usually between 2-4 m. In Fig. 6i, however, the red parts are mainly concentrated in the northeastern part of the lake, because the water depth there is usually between 1 and 2 m, and the water depth in the southwestern flood land is now usually less than 1 m.After the operation of the PLHP (M2), with the rise of the water level in the lake region, the suitable area for the growth of Vallisneria natans is increased greatly, and the variation is proportional to the DWL during the same period. The increase is most obvious in the low-water-level year (2006), followed by the medium water level year (2018), and becomes insignificant in the high-water-level year (2010). Such a trend is consistent with the previously mentioned variation in the water depth.Since the DWL is relatively small during September and October in 2010, the habitat suitability in this period is changed little between M1 and M2 (Fig. 6). The distribution of the habitat suitability is completely the same between Fig. 6c,f, while the difference in the distributions of habitat suitability between Fig. 6i,l is subtle. As the water level in M1 is relatively low during September and October in 2006 and 2018, the suitable area for the growth of Vallisneria natans in M2 is expanded from northeast to southwest under the influence of the PLHP. In addition, the habitat suitability is increased greatly in Wucheng National Nature Reserve and Nanji National Nature Reserve. Before the operation of the PLHP, there are large areas of dry land in the two reserves, and thus the habitat suitability in these dry areas is 0. After the operation of the PLHP, according the previous research, more than 1/3 of area in the two reserves sees apparent increases in water depth. The water depth is increased to 1 ~ 2 m and thus the habitat suitability is increased to 1.0 in the northeastern part of the reserves. In the southwestern part of the reserves, although the increase of water depth is not significant, most of the lake bed has changed its status from being dry to being wet and the habitat suitability is correspondingly increased from 0 to 0.1 ~ 0.2, as shown in Figs. 6d,e,j,k. This means that the two nature reserves will be more suitable for the growth of Vallisneria natans after the operation of the PLHP, and there it will be easier for Siberian Crane to find food here.Changes in habitat area of Vallisneria natansOn the basis of formula (6), we calculate the habitat area from 1st of September to 31st of October in three typical years, as shown in Fig. 7 and Table 3. In 2006, the habitat area is increased greatly, and the impact of the PLHP on habitat area is most evident in October. Compared with M1, the mean monthly habitat area in M2 is increased by 190.92%. Especially around the 10th of October, the increase can reach about 867.79 km2, accounting for about 1/4 of the total area of the lake. Compared with 2006, the increase of habitat area in 2018 is smaller. The mean monthly habitat area in M2 is increased by 57.07% in September and by 145.27% in October. The largest change occurs in late September, with an increase of about 841.43 km2, which is basically the same as in 2006. While in 2010, compared with M1, the habitat area in M2 is completely the same in September and the average increase in October is only 18.07%, indicating that when the water level is relatively high the operation of the PLHP will make little change to the habitat area.Figure 7Variations of habitat areas of Vallisneria natans in M1 (without the PLHP) and M2 (with the PLHP) and the resulting differences (grey columns).Full size imageTable 3 Mean monthly habitat areas of Vallisneria natans and changes between M1 and M2.Full size tableAfter the operation of the PLHP, the habitat area can reach more than 1000 km2 during the most time of September and October in three typical years, accounting for about 1/3 of the total area of the lake region. In other words, the latest official regulatory scheme of the PLHP is beneficial for the growth of Vallisneria natans in September and October. It means that, whether it is a low-water-level year, medium-water-level year or high-water-level year, before Siberian Crane fly to Poyang Lake for winter, Vallisneria natans will occupy large areas of the flooded land under the regulation of the PLHP. It will ensure that Siberian Crane can consume abundant tubers of Vallisneria natans as food in winter, allowing them to better survive and reproduce in the wetlands of Poyang Lake.In this research, the habitat suitability model of Vallisneria natans in Poyang Lake is established based on the previous research of Chen et al.16. This model only considers the effect of water depth, which mainly influences the growth of aquatic plant by changing the degree of light attenuation34. In fact, temperature and flow velocity can also influence the growth of Vallisneria natans according the relevant studies. However, temperature only plays a decisive role in the germination period38 and is rarely considered as an influencing factor during the growth period of Vallisneria natans. While high flow velocity may adversely affect the growth of Vallisneria natans during the seedling period, adult Vallisneria plants have an extensive root–rhizome system and long ribbon-like leaves to prevent them from being torn apart in rapid water39. Vallisneria natans often begin to sprout in March, reaching the tillering stage in June or July in Poyang Lake region. As a consequence, the temperature and flow velocity will have little effect on the growth of Vallisneria natans during the study period in this research (September and October). Therefore, the water depth can be regarded as the main factor affecting the suitability of Vallisneria natans during the mature period.There have been several scholars who have studied the effect of water depth on the growth of Vallisneria natans in other areas. Xiao et al.40 reported that Vallisneria natans grow rapidly with depths of 110–160 cm, while the growth is severely retarded with a depth of 250 cm. Cao et al.34 carried out experiments in turbid water and reported that the water depth of about 130 cm is most suitable for the growth of Vallisneria natans, while higher water depth will be less favorable for the growth. The inconsistency between these findings and the habitat suitability curve proposed by Chen et al.16 may be explained by the climate differences in different regions, as well as the different degrees of turbidity in water. According to the above analysis, the habitat suitability model of Vallisneria natans in this paper is established based on the habitat suitability curve proposed by Chen et al. (2020) as it represents to the growth characteristics of Vallisneria natans in Poyang Lake region.According to the above analysis, the latest regulatory scheme of the PLHP will effectively increase the water level in the lake region and expand the habitat area of Vallisneria natans, especially in low-water-level years. This finding is different from some of the previous research. Zhu et al.15 used the average water depth during the growing period of Vallisneria natans (from March to October) to reflect the availability of this food resource for Siberian Crane. They found that the PLHP had few influences on Vallisneria natans. This was because the PLHP remain completely open from April to August and it takes effect only in March, September and October during the growing period of Vallisneria natans. Therefore, the average water depth during March to October differs little whether with or without the PLHP, which would certainly underestimate the impact of the PLHP. The present study focuses on September and October, and uses the mean monthly water depth to reflect the habitat suitability of Vallisneria natans. In this period, the natural water level is relatively low in M1, and the operation of the PLHP increases the water level and inundates large areas of otherwise dry land. As a result, the habitat areas of Vallisneria natans observe an increase. More

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Study siteThe study area covers the campus of Suranaree University of Technology (SUT) and its surrounding landscape in Muang, Nakhon Ratchasima, Thailand (14.879° N, 102.018° E; Fig. 1). The university campus covers about 11.2 km2, and comprises a matrix of human modified lands interspersed with mixed deciduous forest fragments (at the onset of this study we identified there were 37 mixed deciduous forest fragments on campus, mean = 7.36 ± 1.48 ha, range = 0.45–45.6 ha [note, “±” is used for standard error throughout the text]). More than 15,000 students are enrolled at SUT, and there are numerous multi-story classrooms, laboratory and workshop buildings, residential housing, parking areas, eating and sports facilities, an elementary school, and a large hospital on the university campus. During the first term of the 2019 school year, 7622 students, as well as numerous SUT staff, lived in on-campus residential areas. The landscape surrounding the university is primarily dominated by agriculture, though there are also patches of less-disturbed areas as well as several densely populated villages and suburban housing divisions among the monoculture plots of upland crops (e.g., cassava, maize, and eucalyptus).Figure 1Study site map illustrating the land-use types spanning the area where the Malayan kraits (Bungarus candidus) were tracked in Muang Nakhon Ratchasima, Nakhon Ratchasima province, Thailand. Map created using QGIS v.3.8.2 (https://qgis.org/) in combination with Inkscape v.1.1.0 (https://inkscape.org/).Full size imageThe study site is located within the Korat Plateau region with an altitude range of 205–285 m above sea level. Northeast Thailand has a tropical climate, and the average daily temperature from 1 January 2018 to 31 December 2020 in Muang Nakhon Ratchasima was 28.29 °C, with daily averages ranging from 19.3 to 34.1 °C38. The region receives an average annual rainfall ranging from 1270 to 2000 mm39. There are three distinct seasons in northeast Thailand: cold, wet, and hot, each are classified by annual changes in temperature and rainfall. Cold season is typically between mid-October and mid-February, hot season is generally from mid-February to May, while the highly unpredictable rainfall of the wet season is predominantly concentrated between the months May to October39,40.Due to the representation of agriculture, semi-urban, and suburban areas with patches of more natural areas all within a relatively small area, we determined the university campus provided an ideal setting to examine how land-use features and human activity influence the movements of B. candidus. Additionally, past studies have indicated northeast Thailand hosts the most bites by B. candidus in Thailand29,33, making sites like ours ideal.Study animalsWe opportunistically sampled Malayan kraits captured as a result of notifications from locals and ad-hoc encounters during transit due to low detectability in visual encounter surveys, in addition to those discovered through unstandardized visual encounter surveys. Upon capture, we collected morphometric data, including snout-vent length (SVL), tail length (TL), mass, and sex (Table 1, Supp. Table 1). We measured body lengths with a tape measure, measured body mass with a digital scale, and determined sex via cloacal probing, all while the snakes were anesthetized via inhaling vaporized isoflurane. We then housed individuals with an SVL > 645 mm and mass > 50 g in plastic boxes (with refugia and water) prior to surgical transmitter implantation by a veterinarian from the Nakhon Ratchasima Zoo. We attempted to minimize the time snakes were in captivity awaiting implantation; however, delays arose due to the veterinarian’s availability, the snake being mid-ecdysis, or the snake having a bolus that needed to pass through the digestive tract before implantation (n = 21 implantations, mean = 5.02 ± 0.61 days, range = 0.60–13.02 days). The Nakhon Ratchasima Zoo veterinarian implanted radio transmitters (1.8 g BD-2 or 3.6 g SB-2 Holohil Inc, Carp, Canada) into the coelomic cavity using procedures described by Reinert and Cundall41, while the snake was anesthetized. We assigned each individual an ID according to sex and individual detection number (e.g., M02 = a male was the second B. candidus individual documented during the study). We released the implanted individuals as close as possible to their capture locations (mean = 65.31 m ± 13.7 m, range = 0–226.42 m), though on six occasions we moved individuals ≥ 100 m because the individual came from either residential areas or a busy road (all but one were moved  800 mm; thus, nine of the males were adults and four were juveniles (though two of the males had an SVL > 720 mm, and therefore likely sub-adults). The single telemetered female was an adult.Individual tracking durations varied (mean = 106.46 ± 15.36 days, range = 28.5–222.77 days; Supp. Fig. 1), as many individuals were lost due to unexpected premature transmitter failures (n = 5) or unsuccessful recapture efforts due to individuals sheltering under large buildings as the transmitter reached the end of its battery life (n = 4). We only recorded one confirmed mortality in the study, M01, who was killed by a motorized vehicle when crossing a road (n = 1). Another three individuals were lost due to unknown reasons, which may have been due to premature transmitter failure, mortality, or the animal moving beyond radio signal despite extensive search efforts. Thus, we only successfully recaptured and re-implanted five individuals (M01 once, M02 twice, M07 once, M27 once, and M33 twice). Transmitter batteries generally lasted approximately 90–110 days, so we aimed to replace transmitters after ≥ 90 days of use. At the end of the study, only one individual was successfully recaptured to remove the transmitter.Data collectionWe used very high frequency radio-telemetry to locate each telemetered individual on average every 24.20 h (SE ± 0.41, 0.17–410.0 h; see Supp. Fig. 2 for distribution of tracking time lags). We aimed to locate each individual’s shelter locations once each day during the daylight (06:00–18:00 h); however, we were occasionally (n = 34 days) unable to locate a snake for several consecutive days when we were unable to obtain radio signal due to an individual having moved far away or deep underneath a large structure. There were also a few occasions where we were unable to track snakes due to prolonged and heavy rainfall (n = 4 days), as the moisture damages equipment, or other reasons (n = 4 days). We additionally located snakes nocturnally (18:00–06:00 h) ad hoc and in an attempt to observe nocturnal behaviors and movement pathways when animals were active. We defined fixes as any time a telemetered individual was located, and relocations (i.e., moves) as the occasions where we located an individual > 5 m from its previous known location.Each day we manually honed in on signal via a radio receiver to locate individuals (as described by Amelon et al.42, and recorded locations in Universal Transverse Mercator (UTM; 47 N World Geodetic System 84) coordinate reference system with a handheld global positioning system (GPS) unit (Garmin 64S GPS, Garmin International, Inc., Olathe, Kansas) directly above the sheltered snake. We generally approached within one meter of sheltering snakes during daylight to precisely record shelter locations and identify shelter type. Since we could not visually confirm snake locations, we methodically eliminated all possible locations where the snake could possibly be while at close range with the minimum possible gain on the radio receiver.Telemetered kraits tended to be inactive and sheltering underground during the daylight, thus we were confident that our diurnal location checks would not affect their movements. However, in some cases we resorted to determining an individual’s location via triangulation, where multiple lines cast from different vantage points towards the snake intersect on the snake’s location on the GPS, allowing us to determine the animal’s coordinate location from approximately 10–30 m away. This helped ensure that we recorded locations with greater accuracy when snakes sheltered underneath large buildings, as it allowed us to move away from large structures that hindered the GPS accuracy. This technique was also implemented during some nocturnal location checks when a snake was believed to be active among dense vegetation, in an attempt to prevent disturbance of the animals’ natural behavior. While we did hope to gain visual observations of active individuals during the night, we exercised more caution during nocturnal location checks, typically maintaining a minimum distance of approximately 5 m in attempt to lessen the chances of disturbing an active individual’s behavior. If the animal was active we recorded the animal’s observed behavioral state (i.e., moving, feeding, or foraging). When the radio signal was stable and the individual was not visible, we recorded the animal’s behavior as “sheltering”. We strived for an accuracy of  5 m difference), and land-use type (e.g., mixed deciduous forest, human-settlement, semi-natural area, agriculture, plantation; see Supp. Figs. 3 and 4 for photos of land-use types), behavior (e.g., sheltering, moving, foraging, or feeding), and shelter type (e.g., anthropogenic, burrow, or unknown, note we also recorded if we suspected the shelter to be part of a termite tunnel complex due to a close proximity to a visible termite mound; Supp. Fig. 5).During each location check we recorded the straight-line distance between the current and previous locations (distance moved/step length) with the GPS device. We then used step-lengths to summarize their movements by estimating the mean daily displacement (MDD; the total distance moved divided by the number of days the snake was located) and mean movement distance (MMD; the mean relocation distance, excludes distances ≤ 5). In order to limit biases due to some snakes being located multiple times within a given day/night, we limited our sample for estimating MMD and MDD to only include a single location per day. This was accomplished by manually removing “extra” nocturnal location checks that occurred within the same day, making sure to have all shelter relocations present within the dataset. When calculating MDD, we used the total number of daily location checks rather than the number of days between the individual’s tracking start and stop date since there were some days where individuals were not tracked. We also used the same one location check per day dataset to calculate movement/relocation probabilities and to examine each individual’s MMD, MDD, and relocation probability for the overall tracking duration as well as for each season.When feasible, we positioned a Bushnell (Bushnell Corporation, Overland Park, Kansas) time lapse field camera (Trophy Cam HD Essential E3, Model:119837) with infrared night capability on a tripod spaced 2–5 m from occupied shelter sites. We positioned the cameras so that we may gather photos of the focal snake as it exited the shelter site and/or behaviors exhibited near the shelter. We programmed the cameras using a combined setting, including field scan, which continuously captured one photo every minute, along with a motion sensor setting, which took photos upon movement trigger outside of the regular 1-min intervals.Space use and site fidelityAll analyses and most visualizations were done in R v.4.0.5 using RStudio v.1.4.1106 43,44. We attempted to estimate home ranges for the telemetered B. candidus individuals using autocorrelated kernel density estimates (AKDEs) using R package ctmm v.0.6.045,46 in order to better understand the spatial requirements of B. candidus. However, examination of the variograms revealed that the majority of the variograms had not fully stabilized (i.e., limited evidence of range stability in our sample), and many individuals had extremely low effective sample sizes (21.82 ± 9.75, range = 1.49–135.75; Supp. Table 4). Therefore, we do not report home ranges in this text, as the AKDE estimates would violate the assumption of range residency and either underestimate or misrepresent B. candidus spatial requirements. We also examined the speed estimates resulting from fitted movement models. Resulting variograms and tentative home range estimates are included in a supplementary file for viewing only (Supp. Fig. 6, Supp. Table 4). The original code is from Montaño et al.47.Since our data was not sufficient to estimate home range size for the telemetered B. candidus, we instead used Dynamic Brownian Bridge Movement Models (dBBMMs) with the R package move v.4.0.648 to estimate within study occurrence distributions. We caution readers that these are not home range estimates but instead modeling the potential movement pathways animals could have traversed49. Use of dBBMMs not only allows us to estimate occurrence distributions for each individual, thus helping us better understand the animal’s movement pathways and resource use, but it also allows us to examine movement patterns through dBBMM derived motion variance50,51. We selected a window size of 19 and margin size of 5, to catch short resting periods with the margin, while the window size of 19 is long enough to get a valid estimate of motion variance when the animals exhibit activity/movement. Contours however are somewhat arbitrary; therefore, we used three different contours levels (90%, 95%, 99%) to estimate dBBMM occurrence distributions (using R packages adehabitatHR v.0.4.19, and rgeos v.0.5.5), and show the sensitivity to contour choice52,53.All movement data, either including initial capture locations or beginning with the first location check ~ 24 h post release, was used for production of both the AKDEs and dBBMMs for each individual. We also estimated dBBMM occurrence distributions for each telemetered individual with the exception of M29, which only made three small moves within a burrow complex during the short time he was radio-tracked before transmitter failure.We compared space use estimates to two previously published B. candidus tracking datasets34,36, and one unpublished dataset shared on the Zenodo data repository54, all originating from the Sakaerat Biosphere Reserve (approximately 41 km to the south of our study site): two adult males from within the forested area of the reserve [one tracked every 27.8 ± 0.99 h over a period of 103 days, the other tracked every 38.63 ± 11.2 h over a period of 30.58 days]34,54, and a juvenile male from agriculture on a forest boundary [tracked every 50.19 ± h for 66.91 days]36. The previous studies on B. candidus only tracked the movements of a single individual each, had coarser tracking regimes, and used traditional—fundamentally flawed methods55,56—to estimate space use34,36. Therefore, we ran dBBMMs with these previous datasets using the same window (19) and margin size (5).To quantify site reuse and time spent at sites (residency time) we used recursive analysis with the R package recurse v.1.1.257. We defined each site as a circular area with a radius of 5 m around each unique location (matching the targeted GPS accuracy). Then we calculated each individual’s overall number of relocations, each individual’s total number of relocations to each site, and each individual’s site revisit frequency and residency time at each unique site. Then we plotted revisited locations on a land-use map with space use estimates (95% and 99% dBBMM) in an attempt to help identify and highlight activity centers for telemetered individuals (see Supp. Figs. 7–13). All maps were created using Quantum Geographic Information System (QGIS v.3.8.2).Habitat selectionWe used Integrated Step Selection Function models (ISSF) to examine the influence of land-use features on the movements of B. candidus at both the individual and population levels. We included movement data from all male individuals that used more than one habitat feature in our ISSF analysis. Therefore, we excluded F16 and M29 who both only used settlement habitat. Excluding M29 was justified by the individual having been tracked for the shortest duration (19 days) and had the fewest number of moves (n = 3), thus there were not enough relocations for ISSF models to work effectively. Using modified code from Smith et al.51 that used ISSF with Burmese python radio-telemetry data, we used the package amt v.0.1.458 to run ISSF for each individual, with Euclidean distance to particular land-use features (natural areas, agriculture, settlement, buildings, and roads) to determine association or avoidance of features. Cameron Hodges created all land-use shape files in QGIS by digitizing features from satellite imagery and verified all questionable satellite land-use types via on-ground investigation.The semi-natural areas, plantations, mixed deciduous forest and water bodies (such as irrigation canals and ponds which have densely vegetated edges) were all combined into a single layer of less-disturbed habitats which we refer to as “natural areas”. All feature raster layers were then converted into layers with a gradient of continuous values of Euclidean distances to the land-use features, and were inverted in order to avoid zero-inflation of distance to feature values and to make the resulting model directional effects easier to more intuitive. We were able to generate 200 random steps per each observed step (following Smith et al.51), due to the coarse temporal resolution of manually collected radio-telemetry data (i.e., we were not computational limited when deciding the number of random locations). Higher numbers of random steps are preferable as they can aid in detecting smaller effects and rarer landscape features59.To investigate individual selection, we created nine different models testing for association to habitat features, with one being a null model which solely incorporated step-length and turning angle to predict movement60, five examining land-use features individually (agriculture, buildings, settlement, natural areas, roads), and the other three being multi-factor models. Each model considers distance to a land-use variable, step-length, and turn-angle as an aspect of the model. After running each of the nine models for each individual, we then examined the AIC for each model, point estimates (with lower and upper confidence intervals), and p-values in order to identify the best models for each individual and determine the strongest relationships and trends among the samples. We considered models with ∆ AIC  More

In this picture, taken in February at Copenhagen Zoo, I’m holding a vacuum device equipped with a tiny fan and filter. The devices — we call them air samplers — are designed to collect DNA samples from the air. We deployed three samplers at the zoo: one in a stable with two okapi (Okapia johnstoni) and two duikers, one in a rainforest house and one outside, near an exhibit of animals that live in the African savannah.At best, we had hoped to detect nearby animals in small enclosures — an okapi in the stable, for instance. But as we reported in Current Biology, the devices outperformed our expectations (C. Lynggaard Curr. Biol. 32, 701–707; 2022). They picked up identifiable DNA from 49 vertebrates, including guppies in the rainforest pool, ostriches and giraffes in the savannah area, and even cats and dogs in the park next door. Interestingly, we didn’t get any signal from turtles in the rainforest house. Maybe turtles mostly keep their DNA to themselves.Our analysis ultimately found that the sampler could detect animals from nearly 200 metres away. The giraffe in the picture is standing much closer than is necessary for collection of a sample.Airborne DNA is all around us. Birds release skin cells when they flap their wings. Saliva from all sorts of animals can become airborne. Animals release DNA when they defecate. In November 2021, I received a grant to start a research group whose goal is to collect airborne DNA in nature. This approach could transform conservation biology and species monitoring. We could detect rare animals and get a better understanding of diversity without disturbing an environment.We have so many lines of inquiry in this work. The location of the samplers, the rates of air flow, the time, the best methods for sorting DNA from the sample — we’re still trying to work all of these out. We hadn’t expected that the zoo experiment would ever work, so we’re scrambling to plan the next steps. It’s an exciting time. More

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Fossil dataWe downloaded all fossil occurrences recorded for the order Scleractinia at the species level from the Paleobiology Database (PBDB – paleobiodb.org; accessed on 3 August 2021). This is the most comprehensive repository for palaeontological data in reef corals to date. Due to the nature of the data, no ethics approval was required. To minimize identification issues, we excluded taxa with uncertain generic and species assignments (i.e., classified as aff. and cf.) and only selected species that had accepted names. We also selected the variables classification and palaeoenvironment from the output options to facilitate taxonomic and environmental filters applied in downstream analyses. The full dataset consisted of 24,011 occurrences across 4235 species, spanning over 250 Myr of coral evolution from the Triassic to the present. Although our focus here lies on the Cenozoic, we used the complete fossil dataset (i.e., including all of the occurrences) to have estimates of the diversification dynamics in scleractinian corals throughout the whole timespan of their evolution.Evolutionary ratesWith the full palaeontological dataset, we estimated evolutionary rates through time in scleractinian corals using the Bayesian framework of the program PyRate (v3.0)12,36,37. This program uses fossil occurrence data to calculate the temporal variation in rates of preservation, speciation and extinction, while incorporating multiple sources of uncertainty12. At its core implementation, PyRate jointly estimates the times of origination (Ts) and extinction (Te) for each fossil lineage; the fossilization and sampling parameters that determine preservation rates (q); and the overall rates of speciation (λ) and extinction (μ) through time36. Recently, the program has been upgraded to include a reversible jump Markov Chain Monte Carlo (rjMCMC) algorithm to estimate diversification rate heterogeneity, which provides more accurate and precise estimates than other commonly used methods12. Therefore, despite the inherent bias of the fossil record (i.e., estimates are conditioned on sampled lineages), PyRate is a robust method to quantify speciation and extinction rates, and their respective temporal shifts, from fossil occurrence data.Extant taxa can also be included in the PyRate framework as long as they are also represented in the fossil record. This is done to extend the fossil geologic ranges to the recent times. Hence, the first step in our analysis was to identify which species in our dataset is still alive at the present. To do this, we matched the accepted species names in the PBDB dataset with those from the extant species dataset of Huang et al.38. Subsequently, we split our dataset into eleven independent subsets, with the goal of keeping each subset with an equal number of species. Each data subset included a random selection of species with their respective occurrences, which was enough to calculate Ts and Te (see below). This was done to avoid convergence issues, given the large size of our dataset and the consequent complexity of the model37. For each of our subsets, we generated fifty replicates by resampling the fossil ages from their temporal ranges to account for the uncertainty associated with the age of occurrences. We then used the maximum-likelihood test in PyRate to compare between three models of fossil preservation12: the homogeneous Poisson process (HPP; q is constant through time); the nonhomogeneous Poisson process (NHPP; q varies throughout the lifespan of a species); and the time-variable Poisson process (TPP; q varies across geological epochs). The latter model (TPP) was selected across all of our data subsets (Supplementary Table 2).After selecting the preservation model, we first focused on assessing the estimates of times of origination and extinction in each data subset, rather than using the full dataset to jointly estimate all parameters at once as in the original implementation of PyRate. This further reduced the complexity of the model and allowed for more precise parameter estimates. For each replicate in all of our data subsets, we approximated the posterior distribution of Ts and Te through a 50 million generation run of the rjMCMC algorithm under the TPP, sampling parameters every 40 thousand iterations. At the end of each run, we discarded 20% of the samples as burn-in and assessed chain convergence through the effective sample sizes of posterior parameter estimates, using the software Tracer39 (v1.7.1).From the results of this first set of models, we extracted the median estimates of Ts and Te across replicates, and we merged the estimates from the eleven independent data subsets. This merged data frame contained estimated times of origination and extinction for all coral lineages within our fossil dataset. We then used this merged Ts and Te data frame as input for another rjMCMC chain to finally estimate overall λ and μ through time, by applying the option -d in PyRate. In this option, Ts and Te for all fossil lineages are given as fixed values and, therefore, are not estimated by the model. The chain for this model was run for 100 million generations, sampling parameters at every 40 thousand iterations. Once again, we excluded 20% of the initial samples as burn-in and checked model convergence using Tracer. Finally, we calculated net diversification rates through time by subtracting the post burn-in samples of μ from λ.To explore the taxonomic idiosyncrasies in the evolutionary rates of reef corals, we selected the most abundant families on present-day coral reefs in terms of the number of colonies per area18 (Acroporidae, Agariciidae, Merulinidae, Mussidae, Pocilloporidae, and Poritidae). Altogether, species within these families account for ~40% of the total extant diversity in Scleractinia. These families also account for most of the occurrences in the PBDB fossil dataset (excluding extinct families, which are generally older and had little temporal overlap with extant ones): Acroporidae (1457 occ. in 165 spp.); Agariciidae (722 occ. in 89 spp.); Merulinidae (2464 occ. in 229 spp.); Mussidae (1146 occ. in 100 spp.); Pocilloporidae (615 occ. in 64 spp.); and Poritidae (1149 occ. in 91 spp.). Therefore, from our full dataset, we selected six independent ones encompassing all species in each of the selected families. We also selected only species that are classified as reef-associated within these families, since we were specifically interested in these environments. This selection had a negligible effect on the size of the individual datasets, given that the vast majority of fossil species within these families are reef-associated. In each family, we followed the same modelling steps described above to estimate μ and λ, and diversity trajectories. However, this time it was not necessary to split the datasets into subsets, given that each family has far less occurrences than the full dataset. We started by comparing models of preservation, which showed the TPP as the best supported for all families (Supplementary Table 3). Then we created fifty replicates by resampling fossil ages to accommodate the uncertainty associated with the time of occurrences. For each replicate, we ran the rjMCMC algorithm for 50 million generations under the TPP model, with a sampling frequency of 40 thousand iterations. We discarded initial 20% of the samples as burn-in, and assessed convergence through Tracer. We then combined all replicates, resampling 100 random samples from each replicate to assess the estimates of μ and λ through time for each family. Finally, we extracted diversity trajectories in each family for all of the replicates by applying the -ltt option in PyRate, which generates a table with estimated range-through diversity at every 0.1 Myr. From these trajectories, we calculated the mean difference in diversity (slope in species per 0.1 Myr) between subsequent time samples backwards from the present (i.e., diversity in time t was subtracted from diversity in time t-1) using the diff function in R (v4.0.3).As an alternative to PyRate, we also calculated the diversity dynamics of reef coral fossils using the R package divDyn40, which combines a range of published methods for quantifying fossil diversification rates. Differently from PyRate, the metrics applied in divDyn require that the fossil occurrences are split into discrete time bins. Therefore, these metrics treat the origination and extinction rates as independent parameters in each bin, while PyRate is designed to detect rate heterogeneity through a continuous time setting12. Our goal here, however, was not to compare models but to assess the robustness of our rate patterns and diversity trajectories using alternative methods. We divided our dataset into one-million-year time bins to have enough temporal resolution for rate calculations. To account for the uncertainty in the assignment of fossil ages, we created 50 binned replicates by sampling the age of each occurrence from a random uniform distribution, with bounds defined by the age ranges provided in the PBDB dataset. We then used the divDyn function to calculate the per capita rates of origination and extinction through time (based on the rate equations by Foote41) for all scleractinians (Supplementary Fig. 5a) and for reef-associated acroporids alone (Supplementary Fig. 5b). We also used the same procedure to generate range-through diversity curves for each of the six families selected previously, to compare with the curves generated by PyRate (Fig. 2a). Although the rate results differed between the PyRate (Fig. 1) and the divDyn (Supplementary Fig. 5) approaches, the general patterns remained unchanged. Rates are more volatile through time in divDyn estimates, with larger confidence intervals, which is expected from the metrics applied in the package12,42. Yet, we found the same peaks in extinction for Scleractinia: at the Cretaceous-Paleogene and Eocene-Oligocene boundaries, and at the Pliocene-Pleistocene (Supplementary Fig. 5a). The recent peak in speciation in Acroporidae was also detected, although less strong (Supplementary Fig. 5b). Despite these slight differences in rate estimates, the diversity curves reconstructed through divDyn (Supplementary Fig. 6) mirrored almost exactly the ones found with PyRate (Fig. 2), demonstrating that the overall macroevolutionary trends described herein (Figs. 1 and 2) are robust to methodological choices.Diversity-dependent modelsTo assess the effects of diversity dependency on the evolution of reef coral lineages, we implemented the Multivariate Birth-Death model (MBD)11 within the PyRate framework. This method was first described as the Multiple Clade Diversity Dependence model (MCDD)19, in which rates of speciation and extinction are modelled as having linear correlations with the diversity trajectories of other clades. At its original implementation, the MCDD was developed to assess the effects of negative interactions, where increasing species diversity in one group can suppress speciation rates and/or promote extinction in itself or in other ecologically similar clades19. However, the model also incorporates the possibility of positive interactions, where increasing diversity in one clade can correlate with enhanced rates of speciation or buffered extinction. Through further model developments43, the MCDD was updated to also include a horseshoe prior44 on the diversity-dependence parameters, which helped controlling for overparameterization and enhanced the power of the model to recover true effects43. More recently, this model took its current form as the MBD11, with the additional possibilities of including environmental correlates and setting exponential, rather than just linear, correlations.We first applied the MBD to estimate the diversity-dependent effects of individual extant coral families (i.e., the ones selected in the previous analysis; see Evolutionary rates) in their combined diversity trajectories. From the rjMCMC model results for individual families, we extracted estimates of Ts and Te in each of the fifty replicates and merged them across families. This merged dataset with fifty replicates of Ts and Te was then used as input for the MBD model, where we set the relative diversity trajectories of each individual family as predictors. We also included three key environmental predictors—paleotemperature, sea level and rate of sea-level change—to assess their influence in overall evolutionary rates. The paleotemperature data was obtained from Westerhold et al.45, and consists of global mean temperature estimates for the last 66 million years, averaged across 0.1 Myr time bins. Eustatic sea-level data was downloaded from Miller et al.46, and contains estimates of sea level for the last 100 million years in comparison to present-day levels, also split in ~0.1 Myr time bins. With this dataset, we calculated the average rate of sea-level change per million years, as measured from the absolute difference between subsequent sea-level values backwards in time (i.e., sea level in time t was subtracted from sea level in time t-1). These environmental factors were rescaled between 0 and 1 to maintain all predictors on the same relative scale.Under our MBD model, the speciation and extinction rates of all families combined could change through time and through correlations with the relative diversity of individual families or environmental factors. The strength and directionality (positive or negative) of the correlations are also jointly estimated for each predictor within the model11. We ran both linear and exponential correlation models (see formulas in Lehtonen et al.11) in each of our fifty replicates for 25 million generations, sampling parameters at every 25 thousand iterations. We then compared the linear and exponential models through the posterior harmonic means of their log likelihoods, which supported the exponential one as having a better fit. From the posterior estimates, we summarized the speciation (Fig. 3c) and extinction (Fig. 3d) correlation parameters (i.e., the strength of the effect) by calculating their median and 95% Highest Posterior Density (HPD) interval across replicates. Finally, we also summarized the effect of families on lineage turnover (Fig. 3e), which we conceptualize as the sum of the effects on speciation and extinction.The MBD model also provides posterior samples of the weight of the correlation parameters, which is estimated through the horseshoe prior11. In essence, this prior is able to reliably distinguish correlation parameters that should be considered noise from those that represent a true signal in the data11. The parameterization of the horseshoe prior contains local and global Bayesian shrinkage parameters44 from which shrinkage weights (w) can be calculated (see formulas in Lehtonen et al.11). These shrinkage weights associated with each correlation parameter in the MBD model vary between 0 and 1, with values closer to 0 representing noise and values closer to 1 representing a true signal. Through simulations, it has been shown that values of w  > 0.5 indicate that the correlation parameter in question significantly differs from the background noise, being the correlation positive or negative43. However, as a conservative way to infer the weight of correlation parameters, here we use a value of w  > 0.7 to detect significance. This value was calculated for each diversity-dependence parameter (speciation, extinction and turnover) from the median values drawn from the model posteriors.The spatial distribution of reef-associated taxa varied considerably throughout the Cenozoic, with biodiversity hotspots moving halfway across the globe47. Therefore, the best way to capture this dynamic biogeographic history in reef corals is by analysing global diversity patterns like we did in our main MBD model. However, to assess the robustness of our diversity-dependent results against the influence of geographic scale and site co-occurrences, we repeated all the modelling steps described above with two data subsets. First, we selected only fossil species that have occurrences in the Indo-Pacific Ocean (i.e., 30°W–180°W) within the six families. Second, we excluded sites in which the Acroporidae did not co-occur with the other families. In each of these data subsets, we calculated diversity trajectories and used them as predictors in a separate MBD model. These models had a merged dataset of Ts and Te of all species included in each case (Indo-Pacific and co-occurrences) as a response variable.Finally, we followed the same modelling procedures described above to investigate the diversity-dependent effects in family pairwise analyses. We applied the MBD model to assess the effects of all other families in each individual family at a time, while also estimating correlations with the key environmental predictors. From the rjMCMC model results for individual families, we extracted the fifty replicates of estimated Ts and Te. Each replicate was then used as input for an MBD run using the relative diversity trajectories of each other individual family as predictors, along with the environmental variables. Once again, we ran 25 million generations of the MBD, with a sampling frequency of 25 thousand, using both linear and exponential correlation models in each age replicate. For all families, we found that the exponential model had a better fit. We then summarized the correlation parameters and the shrinkage weights (Supplementary Fig. 7) derived from the exponential models per family by calculating the median and 95% confidence intervals across replicates.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More