Ecology

All Alteromonas strains support long-term survival of Prochlorococcus under N starvationPrevious research showed that Prochlorococcus, and to some extent Synechococcus depend on co-occurring heterotrophic bacteria to survive various types of stress, including nitrogen starvation [33, 34, 42, 43]. At the first encounter between previously axenic Prochlorococcus and Alteromonas (E1), all co-cultures and axenic controls grew exponentially (Fig. 1B, C). However, all axenic cultures showed a rapid and mostly monotonic decrease in fluorescence starting shortly after the cultures stopped growing, reaching levels below the limit of detection after ~20–30 days. None of the axenic Prochlorococcus cultures were able to re-grow when transferred into fresh media after 60 days (Fig. 1C). In contrast, the decline of co-cultures rapidly slowed, and in some cases was interrupted by an extended “plateau” or second growth stage (Fig. 1B). Across multiple experiments, 92% of the co-cultures contained living Prochlorococcus cells for at least 140 days, meaning that they could be revived by transfer into fresh media. Thus, the ability of Alteromonas to support long-term N starvation in Prochlorococcus was conserved in all analyzed strains.Fig. 1: Experimental designs and overview of the dynamics of Prochlorococcus-Alteromonas co-cultures from first encounter and over multiple transfers.A Schematic illustration of the experimental design. One ml from Experiment E1 was transferred into 20 ml fresh media after 100 days, starting experiment E2. Experiment E2 was similarly transferred into fresh media after 140 days, starting experiment E3. Additional experiments replicating these transfers are described in Supplementary Fig. S1. B Overview of the growth curves of the 25 Prochlorococcus-Alteromonas co-cultures over three transfers spanning ~1.2 years (E1, E2 and E3). Results show mean + standard error from biological triplicates, colored by Prochlorococcus strain as in panel D. C Axenic Prochlorococcus grew exponentially in E1 but failed to grow when transferred into fresh media after 60, 100, or 140 days. Axenic Alteromonas cultures were counted after 60 and 100 days, as their growth cannot be monitored sensitively and non-invasively using fluorescence (optical density is low at these cell numbers). D High reproducibility and strain-specific dynamics of the initial contact between Prochlorococcus and Alteromonas strains (E1). Three biological replicates for each mono-culture and co-culture are shown. Note that the Y axis is linear in panels B, C and logarithmic in panel D. Au: arbitrary units.Full size imageIt has previously been shown that Prochlorococcus MIT9313 is initially inhibited by co-culture with Alteromonas HOT1A3, while Prochlorococcus MED4 is not [12, 32]. This “delayed growth” phenotype was observed here too, was specific to MIT9313 (not observed in other Prochlorococcus strains) and occurred with all Alteromonas strains tested (Fig. 1D). MIT9313 belongs to the low-light adapted clade IV (LLIV), which are relatively distant from other Prochlorococcus strains and differ from them in multiple physiological aspects including the structure of their cell wall [44], the use of different (and nitrogen-containing) compatible solutes [45], and the production of multiple peptide secondary metabolites (lanthipeptides, [46, 47]). LLIV cells also have larger genomes, and are predicted to take up a higher diversity of organic compounds such as sugars and amino acids [48]. It is intriguing that specifically this strain, which has higher predicted metabolic and regulatory flexibilities [49], is the only one initially inhibited in co-culture with Alteromonas.Differences in co-culture phenotype are related to Prochlorococcus and not Alteromonas strains and occur primarily during the decline stageWhile co-culture with all Alteromonas strains had a major effect on Prochlorococcus viability after long-term starvation, there was no significant effect of co-culture on traditional metrics of growth such as maximal growth rate, maximal fluorescence, and lag phase (with the exception of the previously described inhibition of MIT9313; Fig. 2A–C). However, a visual inspection of the growth curves suggested subtle yet consistent differences in the shape of the growth curve, and especially the decline phase, between the different Prochlorococcus strains in the co-cultures (Fig. 1D). To test this, we used the growth curves as input for a principal component analysis (PCA), revealing that the growth curves from each Prochlorococcus strain clustered together, regardless of which Alteromonas strain they were co-cultured with (Fig. 2D). The growth curves of all high-light adapted strains (MED4, MIT9312, and MIT0604) were relatively similar, the low-light I strain NATL2A was somewhat distinct, and the low-light IV strain MIT9313 was a clear outlier (Fig. 2D), consistent with this strain being the only one initially inhibited in all co-cultures. Random forest classification supported the observation that the growth curve shapes were affected more by the Prochlorococcus rather than Alteromonas strains, and also confirmed the visual observation that most of the features differentiating between Prochlorococcus strains occurred during culture decline (random forest is a supervised machine learning algorithm explained in more detail in Supplementary Text S2; see also Supplementary Fig. S2). Thus, co-culture with Alteromonas affects the decline stage of Prochlorococcus in co-culture in a way that differs between Prochlorococcus but not Alteromonas strains.Fig. 2: Growth analysis and principal component analysis (PCA) of the growth curves from all co-cultures during 140 days of E1.A Growth rate, B Maximum fluorescence, and C duration of lag phase during experiment E1. Box-plots represent mean and 75th percentile of co-cultures, circles represent measurements of individual cultures of the axenic controls. The only significant difference between axenic and co-cultures is in the length of the lag phase for MIT9313 (Bonferroni corrected ANOVA, p  More

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Chromatographic separation and mass spectrometric optimizationTo obtain the best monitoring conditions for each compound, a 0.5 mg/L mixed standard solution of 11 pesticides was mixed with the mobile phase through a syringe pump and then injected into the mass spectrometer for tuning. The precursor ion of the compound to be tested was determined by the primary mass spectrometry scan under ESI+ and ESI- modes, and then the product ion was scanned by the secondary mass spectrometry. Two groups of ion pairs with the best sensitivity were selected for detection; one group was used for quantification, and another, for qualitative analysis. The optimization results showed high sensitivity of all the 11 pesticides under the ESI+ mode. Among them, abamectin (B1a), β-cypermethrin, deltamethrin, fenpropathrin, and bifenthrin were [M + NH4]+, and other compounds were [M + H]+. MS parameters of 11 pesticides are mentioned in Table S2.Formic acid and ammonium acetate are commonly used reagents to enhance the ionization of target compounds [M + H]+ and [M + NH4]+ under the ESI+ mode, and they can effectively improve the peak pattern, making the peak sharper and more symmetrical; therefore, they need to be added during gradient elution38. To improve work efficiency, it is necessary to separate and complete the monitoring of 11 pesticides in the shortest possible time; therefore, we selected two different types of chromatographic columns (ACQUITY UPLC HSS C18 and ACQUITY UPLC HSS T3) and three different mobile phases (Ι: 0.1% formic acid aqueous solution—ACN, II: 0.05% formic acid aqueous solution—ACN, and III: 0.1% formic acid/5 mmol/L ammonium acetate aqueous solution—ACN) for optimization experiments. We observed that when using the HSS T3 chromatographic column, β-cypermethrin, deltamethrin, fenpropathrin, and bifenthrin did not show a good retention effect under the three mobile phase systems, and there was substantial tailing of the chromatographic peak. The shape of the chromatographic peak and sensitivity of the target compound were used as evaluation indicators. Compared with Ι and II, mobile phase III produced better sensitivity for all target compounds (Fig. 1), with sharper and more symmetrical peaks of β-cypermethrin, deltamethrin, fenpropathrin, and bifenthrin. This may be because the addition of 5 mmol/L ammonium acetate improved the retention performance of the HSS C18 chromatography columns without affecting the ionization efficiency of all target compounds. In summary, we selected the HSS C18 column for chromatographic separation and used 0.1% formic acid/5 mmol/L ammonium acetate aqueous solution—ACN as the mobile phase to further optimize the gradient elution procedure and effectively separate and detect all the target compounds within 8 min.Figure 1When using HSS C18, the peak areas of 11 pesticides in three different mobile phases.Full size imageOptimization of purification materialsThe flesh of apricot contains sugar, protein, calcium, phosphorus, carotene, thiamine, riboflavin, niacin, and vitamin C. Due to these diverse impurities, the analysis of the sample matrix becomes highly complex. Therefore, these impurities need to be removed from the matrix samples before analysis. Currently, PSA, C18, and MWCNTs are widely used to adsorb to the fruit substrate39. PSA has a strong adsorption capacity for metal ions, fatty acids, sugars, and fat-soluble pigments, C18 has a strong adsorption capacity for non-polar impurities (such as fat, sterol, and volatile oil), while MWCNTs have a strong adsorption capacity for pigments, which can effectively remove chlorophyll, lutein, and carotene. However, C18 and MWCNTs can also simultaneously adsorb pesticides, resulting in poor recovery. Nano-ZrO2 has a large specific surface area and good adsorption stability and has recently been used to purify substrates. It can selectively remove fats and pigments from samples compared to conventional C18 fillers.In the current study, different purification materials were combined for the analysis of 11 pesticide residues and to propose the best purification strategy in the pretreatment of apricot samples. As displayed in Fig. 2, the average recovery of 11 pesticides in the apricot was higher using the C18/nano-ZrO2/MWCNTs than other combinations. Nano-ZrO2 showed better adsorption than PSA in purifying fatty acids, organic acids, polar pigments, and sugars in apricot, owing to its larger specific surface area, better adsorption capacity, and stability. To conclude, the combination of 10 mg C18, 30 mg nano-ZrO2, and 5 mg MWCNTs demonstrated the best recovery for 11 pesticides, with recovery in the range of 72% to 114%, at a pesticide spiking level of 0.01 mg/kg. In summary, we finally determined that among the tested combinations, C18/nano-ZrO2/MWCNTs (10 mg/ 30 mg/5 mg) is the best purification combination for the pre-treatment of apricot samples.Figure 2The recoveries of 11 pesticides in apricot matrix under different scavenger combinations (2–1 C18/nano-ZrO2/MWCNTs, 2–2 PSA/C18/MWCNTs, 2–3 nano-ZrO2/PSA/MWCNTs; 0.01 mg/kg, n = 3).Full size imageLinearity, matrix effects, limit of detection and limit of quantificationThe standard curve obtained from the standard working solutions of 11 pesticides and the calibration curve from blank apricot matrix spiked with 11 pesticides showed good linearity (0.001, 0.005, 0.01, 0.05, 0.1, and 0.5 mg/L), with R2 ≥ 0.9959 for all tested samples (Table 1).Table 1 The standard curves, R2 and MEs of 11 pesticides in apricot.Full size tableTo evaluate MEs, the slopes of matching 11 pesticide standards with solvent and apricot matrix were calculated at the same concentration. According to the derived slope of the matrix-matched calibration curve, MEs of 11 pesticides in apricot were between 89 and 113% (Table 1), well within the range of 80% to 120%, indicating that the MEs could be ignored. It also suggests that the current pre-treatment method has a good purification effect and eliminates the matrix effect very well, laying a robust foundation for the subsequent step of quantitative analysis of samples. We next used the standard solution curve to quantify the 11 pesticide residues in apricot.The LOD refers to the minimum concentration or minimum amount of a component to be tested that can be detected from a test sample under a given confidence level by an analytical method. Its physical meaning is the amount of the measured component when the signal is 3 times the standard deviation (S = 3σ) of the reagent blank signal (background signal). Sometimes it also refers to the amount of the measured component corresponding to when the signal is three times the background signal generated by the reagent blank (S = 3 N). The LOQ refers to the minimum amount of the analyte in the sample that can be quantitatively determined, and the determination result should have a certain accuracy40. The LOQ reflects whether the analytical method has the sensitive quantitative detection ability. The LOQ is the lowest validated level with sufficient recovery and precision, which was estimated to be 0.001 mg/L, while the LOD is the lowest calibration level, which was 2 µg/kg, according to SANTE/12,682/2020.Accuracy and precisionIn the matrix, 11 pesticides were spiked at four levels (0.002, 0.02, 0.1, and 1 mg/kg), and for each spiked sample, there were six replicates. The recoveries of 11 pesticides in apricot at all levels ranged between 72 and 119%. The inter- and intra-level relative standard deviations (RSDs, %) of 11 pesticides in apricot were  More

The richness of Melanesian FrogsApproximately 7.2% (534 out of 7404) of Earth’s recognised frog species occur in Melanesia, a region comprising < 0.7% of the world’s land area. Frog richness in Melanesia, and especially on New Guinea and nearby land-bridge islands (471 species), is higher than in any other tropical insular region (Fig. 1a). New Melanesian frog species have been described at an average rate of nearly 13 species/year since 2000, and the recognised frog fauna has grown by > 50% in that timeframe (Fig. 1b). The authorship of new species has been concentrated, with six authors featuring on 20 or more descriptions since 2000, and one or more of these six authors on every species description since 2000. A small number of species descriptions has included genetic data (31 species), although a higher number of Melanesian frog species have at least one sequence available on GenBank (~38%, or approximately 200 species). This taxonomic work has revealed or emphasised many evolutionary novelties (Fig. 2): multiple apparently independent derivations of extremely miniaturised vertebrates22,23,24, including some of the world’s smallest known tetrapods23,25,26; multiple derivations of complex parental care in different genera27,28; frequent evolutionary shifts between terrestrial, arboreal and scansorial lifecycles22,29; the most extreme sexual size dimorphism yet documented in anurans30; drastic ontogenetic colour change31; a radiation of canopy-dwelling treefrogs32 that show extensive finger webbing and parachuting behaviour convergent with unrelated frog lineages in Asia and the Neotropics; and treefrogs with erectile noses33,34. Taxonomic work has also elucidated novel concentrations of range-restricted endemic taxa, especially in the Milne Bay Region at the far eastern edge of New Guinea21.Fig. 1: Temporal trends in the documentation of the Melanesian frog fauna.a Species accumulation curves for species-rich ( >100 species) insular frog biotas (Species lists from AmphibiaWeb as of 1 October 2021). Separate accumulation curves are given for the entire fauna of Melanesia (including New Guinea), and the fauna of New Guinea and nearby predominantly land-bridge islands. b Species accumulation curve for frogs within Melanesia. Bar at end indicates predicted number of species in each major family based on known, but as yet undescribed candidate species.Full size imageFig. 2: Melanesian frog species described within the last 15 years illustrating the ecological and morphological diversity of the fauna.a Paedophryne titan and b Choerophryne gracilirostris – examples of lineages that have undergone convergent minaturisation; c Choerophryne alpestris – a fossorial species within a largely scansorial lineage; d Xenorhina macrodisca – scansorial species within a largely fossorial lineage; e Cornufer custos and f Oreophryne oviprotector – independent derivations of complex parental care; g Litoria pallidofemora – extensive digital webbing for parachuting; and h Litoria pinocchio – sexually dimorphic and erectile rostral spikes. Photographs F. Kraus (a), S. Richards (b–g), and courtesy of T. Laman (h).Full size imageFrog species richness in Melanesia is highly concentrated into just three families, with Pelodryadidae (137 recognised species, estimated ~200) and especially Microhylidae (317 species, estimated >400) dominating. Melanesian Pelodryadidae are phylogenetically interdigitated with relatives in Australia, suggesting multiple dispersals between the two regions35. In contrast, ancestors of the direct-developing microhylids colonised Melanesia from Asia via trans-marine dispersal likely only once36, radiated across open ecological niches37, and are now the most species-rich insular radiation of frogs in the world. The third major family comprises an ecologically diverse radiation of the direct-developing Ceratobatrachidae (57 species, estimated 66) largely associated with island-arc terranes of East Melanesia and the Philippines, indicating a long history of insular diversification and trans-marine dispersal38. The predominance of direct-developing frogs in Melanesia (~70% of species) mirrors insular faunas in Madagascar (~34%), Sri Lanka (~67%) and the Greater Antilles (~87%). The other four frog families in Melanesia are all relatively species poor (2, 3, 4, and 13 species) (Fig. 1a), centred in New Guinea, and include lineages originating in Asia (Ranidae, Dicroglossidae) or Australia (Myobatrachidae, Limnodynastidae).The described diversity of Melanesian amphibian species remains an underestimate. Survey work and investigation of museum collections by the co-authors identified ~190 additional candidate species distributed across 16 different genera, mostly from Papua New Guinea, suggesting a total richness of over 700 frog species (Fig. 1a, Supplementary Table 1). This estimated percentage of undescribed diversity (~25%) mirrors estimates for the New Guinean flora (~18–22%)7. The majority of candidate species are concentrated in the two most diverse families (Microhylidae and Pelodryadidae), although genetic, morphological, and acoustic evidence indicate the diversity of Melanesian Ranidae is also underestimated (S. Richards and F. Kraus pers. obs.). Most material documenting candidate species has been collected in the last 20 years, and the vast majority is from Papua New Guinea (Supplementary Fig. 1). There is some suggestion of a slowing in the rate of candidate species discovery in the last decade (Supplementary Fig. 2); however, several of the most active field workers in this region have ceased survey work in recent years, which likely accounts for much of this decline. The pervasiveness of complexes of morphologically and/or acoustically cryptic taxa is poorly understood; survey work continues to reveal novelties, and large areas of the region remain unsurveyed or undersampled. In particular, comparisons of area-to-diversity ratios between the better-known eastern portion of New Guinea (Papua New Guinea) with the poorly surveyed western (Indonesian) portion of the island further suggest that, even with candidate species included, diversity in the latter region may be underestimated by as much as 50% (Supplementary Methods and Results, Supplementary Table 2). These trends and patterns all indicate that ~ 700 species is a very conservative minimum estimate of total diversity and support analyses in other taxa showing Melanesia remains a hotspot of unrecognised diversity39,40.Endemism and distributional patternsThe Melanesian frog fauna is highly endemic (97.2%), with tiny proportions of species shared with Australia (2.4%) or with islands farther west in Indonesia (0.6%), indicating that Australia and Melanesia are discrete centres of frog diversification, despite periodic connection via land bridges through the late Tertiary41. The vast majority of Melanesian frog species (471) occur on New Guinea and nearby land-bridge islands (Raja Ampats, Japen and the Milne Bay islands). In comparison, the frog fauna of the much smaller region of Maluku is depauperate (16 species, of which nine are endemic) but also almost certainly underestimated (e.g., there are no Microhylidae recorded from Buru). Most taxa from Maluku are congeneric (and several conspecific) with lineages centred on New Guinea, supporting the biogeographic clustering of Maluku’s amphibians with the main island of New Guinea. In contrast, the frog fauna of East Melanesia is more diverse and highly endemic and dominated by an ecologically diverse radiation of a different family (Ceratobatrachidae) with only four (all pelodryadid treefrogs) out of 56 species shared with nearby New Guinea. East Melanesia and New Guinea appear to be discrete and long-isolated centres of diversification, as expected from their independent geological histories42.Melanesia spans five countries, and this has possibly to some degree masked the exceptional species diversity of the overall region. Papua New Guinea has the highest number of species (398) and endemic species (318). This likely reflects some combination of its slightly larger area (when islands to the north are included), more diverse geological origins, and greater inventory work than seen in neighbouring regions of Indonesia7. Papua, West Papua and Maluku (Indonesia) have many fewer documented species (197), of which a majority (134) is endemic. The boundary between Papua and Papua New Guinea is visible in species-richness maps (Fig. 3a), with lower diversity to the west, indicating that the distribution and diversity of frogs in Indonesia remain less documented in science. The frog faunas of the Solomon Islands (21 species) and Fiji (two species) are more depauperate but include a significant endemic or near-endemic component, whereas the geographically intervening islands of Vanuatu support no native frogs.Fig. 3: Frog species richness in Melanesia based on IUCN distributional maps for all species described by 2019.a All species; b Ceratobatrachidae; c Microhylidae; d Pelodryadidae. Areas of highest estimated diversity correspond to mountain ranges in central and northern New Guinea. The boundaries between Maluku, New Guinea and East Melanesia are indicated. Fiji has only two frog species and is geographically distant from other areas of Melanesia inhabited by frogs and is not visble on this map.Full size imageBased on distribution maps generated for all species recognised by 31 August 2019, the highest regional alpha diversity of frogs occurs along the Central Cordillera of New Guinea (especially in Papua New Guinea) and around the higher mountain ranges along the north coast of Papua New Guinea (Fig. 3a). These centres of diversity correlate with extensive areas of hill and montane forest and broadly correspond with elevational species-richness patterns for mammals and birds in Melanesia43 and for many other taxa elsewhere in the tropics44,45. Large areas of montane forest with lower species richness along the northern versant of the Central Cordillera in Papua New Guinea and in mountain ranges across Papua certainly reflect inadequate sampling. The ceratobatrachid-dominated frog fauna of East Melanesia is richest in Bougainville (Fig. 3d), with attenuating richness towards the west and especially to the east. The two most speciose families both show alpha diversity peaks in mountainous areas of central New Guinea (Fig. 3c–d). In contrast, microhylids are largely absent from the seasonally dry woodlands of the Trans-Fly region in southern New Guinea and exhibit high diversity in northern New Guinea, whereas pelodryadids are much more speciose in the lowlands of southern New Guinea than northern New Guinea. These broad trends may have both ecological (sensitivity of direct-developing microhylids to dry conditions) and historical (Australia as a centre of origin for savanna-adapted Pelodrydidae) underpinnings.The historical and contemporary factors underpinning high frog species diversity in New Guinea remain largely unstudied, especially when compared to other species-rich insular amphibian faunas such as Madagascar46 or the Greater Antilles47. When compared to some areas of the Neotropics, alpha and beta diversities of frogs in lowland forests in the basins of the Sepik and Ramu rivers in New Guinea are unremarkable48. However, the Milne Bay Region has exceptionally high levels of endemism21, so species turnover will be higher in this area. Extent-of-occurrence estimates derived from IUCN maps indicate that direct-developing microhylids have smaller mean and median range sizes than all other families of frogs in Melanesia (Supplementary Table 3). Microhylidae also dominate anuran species diversity in Milne Bay21 and many mountain areas where standing water is very limited49. These data suggest that, as with some areas in the Neotropics50, high beta diversity in lineages with direct development is a key factor underpinning amphibian megadiversity in Melanesia. To address these questions further, synthetic analyses are required to better quantify the extent to which regional megadiversity in Melanesia reflects high community diversity versus species turnover, how elevation and insularity moderates these two parameters, and to what extent emergent patterns may differ from diverse frog communities in other regions such as the Neotropics.The conservation status of Melanesian FrogsThe frog fauna of Melanesia is currently less threatened but more Data Deficient than other comparable insular regions (Fig. 4a). The vast majority of Melanesian frogs are categorised as Least Concern (68%) or Data Deficient (24%). Thirty-one species (6%, or 8% if Data Deficient taxa are excluded) are threatened (Critically Endangered, Endangered, Vulnerable) (Supplementary Table 4), and eight species are considered Near Threatened. No species are assessed as Extinct or Extinct in the Wild. Since the first Global Amphibian Assessment in 2004, the number of Melanesian frog species has grown by 44%, and nearly 60% of the 31 Melanesian frog species now considered threatened were described after 2004 (Fig. 1a). Only one change in status between 2004 and 2019 was considered genuine (Cophixalus sphagnicola), due to the emerging threat of a newly opened mine. All other status changes (for 116 taxa) reflect better information on distribution or changed assessment protocols (Supplementary Table 5). Applying stricter criteria for use of the Data Deficient category in the 2019 IUCN assessment reduced the number of Data Deficient species when compared to 2004 (125 versus 197), but Melanesia still has a higher percentage of Data Deficient taxa than other species-rich tropical insular faunas (Fig. 4a).Fig. 4: The conservation status of Melanesian frogs.a Comparison of number of species in each IUCN threat category across Melanesia, other diverse insular regions, and the nearby continent of Australia. Melanesia has a proportionally low number of threatened taxa but high number of Data Deficient taxa (EX Extinct, CR Critically Endangered, EN Endangered, VU Vulnerable, NT Near Threatened, DD Data Deficient, LC Least Concern, NE Not Evaluated); b Slopes around Mt Simpson, Milne Bay Province, a hotspot of threatened frog diversity due to forest loss through conversion to anthropogenic grasslands; c Choerophryne sanguinopicta from Mt Simpson (Critically Endangered); d Oreophryne ezra from Rossel Island (Critically Endangered) and; e Cornufer citrinospilus from New Britain (Vulnerable). Photographs F. Kraus (b–d), S. Richards (e).Full size imageAll Critically Endangered and Endangered—and most of the Vulnerable—species were listed because of their small extent of occurrence and on-going decline in habitat area and/or quality (criteria B1ab(iii)) (Supplementary Table 6). The key threatening processes were typically forest disturbance or loss due to conversion to plantations or gardens, repeated burning, or mining (Fig. 4b–c). Only two insular species with very localised montane distributions were considered threatened by climatic disturbance and/or climate change alone (Cornufer citrinospilus and Oreophryne ezra) (Fig. 4d–e). No species were currently declining from pathogens, and in particular Batrachochytrium dendrobatidis (Bd), which remains undetected in Melaneisa51. However, the introduction and establishment of Bd has been identified as a severe threat for well over one hundred taxa52, especially for montane pelodryadid treefrogs, a group that has been devastated by this disease in parts of Australia.Although much of New Guinea has historically been considered a ‘wilderness area’ with comparatively little human impact53, the distributions of threatened taxa also highlight areas of conservation concern wherein range-restricted (often single-island endemic) taxa overlap with extensive and increasing anthropogenic impacts (Fig. 5a–b). Nearly half the species identified as threatened (13) are restricted to a recently delineated dramatic centre of herpetofaunal endemism in the Milne Bay Region at the eastern tip of Papua New Guinea21. Three clusters of small-range endemics in this region (all documented in the last two decades) present immediate conservation issues. The first is Mount Simpson, where six microhylids (four named, two awaiting description) with highly restricted ranges are threatened by habitat loss, especially repeated burning and associated conversion of forest to grassland (Fig. 4b). The second is Woodlark Island, where the status of seven endemic microhylids (six named, one undescribed) is likely to worsen rapidly if current, approved proposals to convert large areas of primary forest to oil-palm plantation and/or gold mines proceed21. Finally, Misima Island is home to four endemic microhylids (two considered threatened) with ranges that overlap areas disturbed by mining and forest loss21. Other regions with multiple overlapping threatened taxa are the Adelbert Mountains in Morobe Province (two species), New Britain (two lowland species and one highland species), and Greater Bukida in the Solomon Islands (three lowland species). These clusters of narrow-range taxa highlight important—and in most cases largely overlooked—conservation priorities for Melanesian frogs (and likely other taxa as well21,54). The high percentage of Data Deficient species and low level of survey effort in many areas (especially Papua and West Papua Provinces, Indonesia) also raise the possibility that other threatened hotspots remain overlooked. One area of particular concern may be the island of Biak in Indonesia, which has lost much of its primary vegetation but is home to at least three endemic frogs (one Data Deficient, two Least Concern).Fig. 5: The distribution of threatened frogs in Melanesia.a The estimated distribution of all 31 Melanesian frog species considered Critically Endangered, Endangered or Vulnerable at the end of 2019. Distributional areas are not colour coded by the number of threatened taxa. b Close up of the Milne Bay endemism hotspot. Distributional areas are colour coded by number of taxa, with darker tones indicating more taxa. In both a and b upland areas or islands where the distributions of two or more threatened species overlap are labelled and the number of threatened taxa are indicated in parentheses. Background maps uses the Shuttle Radar Topography Mission (SRTM) 30-meter digital elevation model, accessed from USGS Earth Explorer (https://earthexplorer.usgs.gov/).Full size imageUnderstanding and conserving a megadiverse biotaThe Melanesian flora and frog fauna are both now shown to be megadiverse and highly endemic, yet both also remain poorly known with large areas under-surveyed. An updated comprehensive assessment of threats and taxonomic trends across the frog fauna presented here further highlights that the biota of Melanesia remains relatively intact and less threatened when compared to other biodiverse insular regions. However, a large proportion of the fauna remains Data Deficient or undescribed, and key hotspots of endemism have been overlooked and are increasingly threatened. In both plants and anurans much scientific knowledge of Melanesia’s biota has also been contributed by a relatively small number of productive, but later-career researchers based outside of Melanesia7.Further documenting and conserving the exceptional diversity of Melanesia presents a suite of challenges and opportunities. Recommendations to enable improved documentation of plant megadiversity in Melanesia7 centre around training, capacity-building and support for taxonomy in Melanesia and globally, improving access to specimen collections and diagnostic resources, and ongoing support for survey and collecting within Melanesia. These recommendations apply equally to amphibians. However, addressing these challenges is tempered by the limited career opportunities available to ecologists and taxonomists (both in developed, but especially in developing countries), the variable quality of scientific infrastructure that exists across the region, and the high cost of doing fieldwork in remote areas with limited logistical infrastructure. In the context of these challenges, we hereby focus on suggesting some short-term key priorities and opportunities to build capacity for understanding and conserving frog biodiversity in Melanesia.First, over the last twenty years opportunities to employ Melanesian nationals in survey, monitoring and outreach work have been (and will continue to be) generated predominantly by NGOs, universities and large-scale extractive projects, for example through recent work in the gas fields of the Papua New Guinea Highlands49. While there are diverse perspectives on extractive industries, monitoring and survey work associated with large development projects are a key source of funds to provide training to enable Melanesians to undertake biodiversity work within the region. A key driver of this is strong environmental legislation required by some governments and major lending agencies, in particular the International Finance Corporation under Performance Standard 655. These requirements need to be maintained, enforced and, where possible, exceeded.To further support fieldwork by national scientists there is a need for more readily accessible identification resources for Melanesian researchers, land-owners and managers. An up-to-date comprehensive identification guide to the frog fauna of the whole region would assist and promote taxonomic, ecological and conservation research. However, for many Melanesians, small, regionally focused guides are more usable. These have already been produced for several areas (Supplementary References), providing a model that can be updated and transferred to other regions. Mobile phones are widely used throughout Melanesia, so app- and online-based identification resources may become increasingly accessible. Smartphone-friendly citizen science platforms like iNaturalist56 or even Facebook groups57 also provide potentially powerful resources through which locally collected data can be captured, vetted and disseminated, although their use is currently limited in Melanesia due to patchy internet coverage in many areas. Working with and supporting people from Melanesia to explore and increase the use of these resources could help to ensure longer-term preservation and accessibility of species records and associated data.The latest IUCN assessment for Melanesian frogs also highlights how taxonomic and conservation knowledge is accumulating rapidly. The key geographic areas of threat identified in our study were largely invisible to assessments made less than two decades ago (in 2004) both because the relevant taxonomic work had not been done, and because the situation in Melanesia is changing rapidly. To keep track of these rapid changes it is critical for workers in the region to work together to synthesise and collate new taxonomic, distributional and conservation data. Indeed, since the 2019 IUCN assessment over 20 additional species of Melanesian frogs have been described, and their conservation status should be assessed as a matter of urgency. Preliminary conservation assessments against IUCN criteria are increasingly being included in descriptions, and this trend should be supported and encouraged. More Melanesian nationals need to be involved in conservation assessment processes. Updated comprehensive conservation assessments of other vertebrate groups will also identify complementarity of conservation priorities among taxa in the Melanesian region.Patterns of distribution and threat suggest some geographic priority areas for documenting the diversity of amphibians (and potentially other low-vagility taxa) in Melanesia. First, work in eastern New Guinea has allowed the delineation of geographically localised clusters of threatened taxa that have until now gone unnoticed, perhaps in part because of the designation of much of Melanesia as a sparsely populated and comparatively undisturbed ‘wilderness’ area21. Most threatened frog taxa in these regions are associated with small islands or isolated ‘sky island’ mountains. The degree to which other taxa show endemism in these areas is poorly known. The biotas of potentially comparable islands in Indonesia such as the Raja Ampat Islands, Geelvink Bay and southern Maluku, also remain poorly known, suggesting additional priority areas for survey, taxonomic investigation and conservation assessment. Second, mid-elevation areas show highest alpha diversity, but large areas of this habitat, especially along the northern slopes of the Central Cordillera, remain poorly surveyed. The frog pathogen Bd has devastated montane communities of two Australian frog families that also occur in montane New Guinea (Myobatrachidae and Pelodryadidae)52. In the unfortunate event that Bd colonised New Guinea a wave of rapid declines and extinctions would likely follow52, so a strong baseline of information on montane species diversity, distributions and population status is critical for detecting these impacts. More

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Sites overview and characteristicsThis study focused on three regions located in subantarctic, arctic, and subarctic latitudes. The respective latitudinal and longitudinal ranges covered in this study were: 54.95 to 52.08 °S, and 72.03 to 67.34 °W in Patagonia; 67.44 to 67.54 °N, and 86.59 to 86.71 °E in Siberia; 63.21 to 68.63 °N, and −150.79 to −145.98 °W in Alaska (Figs. 1 and 2). The exact coordinates for each sample were included in the submitted dataset. The field campaigns were conducted in 2016, during the summer for each respective region: January-February in Chilean Patagonia, June-July in Alaska and July-August in Siberia.Fig. 1Location of the three areas included in this study (panel a). The permafrost state and the number of sites and samples per region is indicated for each area. General views of 5 sites are provided as examples (b–f). Panel B provides a large view of the ecosystem surrounding the wetland ALP2 (Alaska, exact location indicated by the white circle). Lake PCL1 (panel c) is representative of the lakes on Navarino island (Chilean Patagonia). The glacial lake SIL2 is shown in panel d. At site SIP5, the hollow at first plan is surrounded by palsa (hummock, second plan), characterized by dark organic matter and lichen vegetation (panel e). The PPP3 peatland shown in panel f is dominated by Sphagnum magellanicum, like most peatlands in the area.Full size imageFig. 2Maps of sampling sites in Patagonia, Alaska and Siberia, indicating the ecosystem type (lake, wetland, soil). The tables show the complete- (in white) and the partial- (in grey) characterization sites. The exact coordinates of each sample are provided in the data record (See data records section).Full size imageFor every site included in the present study, a set of nine qualitative environmental and/or ecological site-scale descriptors was selected and adapted from ENVO Environment Ontology40, which included for example permafrost state, biome, environmental feature and vegetation type (Table 1, Fig. 3). Permafrost state was obtained from the NSIDC permafrost map41. The biome, large-scale descriptor based on climate and vegetation criteria, was derived from Olson et al.42. Temperate forest, boreal forest, and tundra biomes were included. The environmental features that were representative for the three regions were considered: lakes, wetlands, broadleaf/coniferous/mixed forest soils, grassland, tundra, and palsa. All the metadata was included in the submitted dataset. Table 2 summarizes the main types of sampled ecosystems and their main characteristics in the three regions, while Supplementary Table S1 provides the details of each sampling site.Table 1 Overview of the dataset contained in Mimarks sheet.Full size tableFig. 3Description of the qualitative environmental/ecological descriptors used to describe every sample, derived from ENVO Environment Ontology40.Full size imageTable 2 Main types of sampled ecosystems in the three studied regions.Full size tableIn Alaska, the studied area ranged from the Alaska Range and Fairbanks area (interior, continental climate, 63–65°N, discontinuous permafrost) up to Toolik Field Station (North Slope, arctic climate, 66–69°N, continuous permafrost; Fig. 2). The physiochemistry and CH4 emissions of lakes ALL1 (Killarney lake), ALL2 (Otto lake), ALL3 (Nutella lake), and ALL4 (Goldstream lake) were previously characterized35. A number of heterogeneous soil and wetland samples were collected around the studied Alaskan lakes and/or from monitored sites, as detailed in Supplementary Table S1. In the Alaska Range and Fairbanks area, soils were mostly covered by mixed or taiga forests, alpine tundra, and bogs or fens wetlands. In the norther Brooks Ranges mountain system, the landscape was piedmont hills with a predominant soil of porous organic peat underlain by silt and glacial till, all in a permafrost state, characterized mainly by Sphagnum and Eriophorum vegetation, as well as dwarf shrubs.In Siberia, the studied area was located in the discontinuous permafrost region surrounding Igarka, on the eastern bank of the Yenisei River (Fig. 2). This region was mainly covered by forest, dominated by larch (Larix Siberica), birch (Betula Pendula), and Siberian pine (Pinus Siberica), and palsa landscapes (frozen peat mounts), the latter being dominated by moss, lichens, Labrador tea and dwarf birch. In degraded areas, thermokarst bogs were dominated by Sphagnum spp. and Eriophorum spp. Land cover was an indicator of permafrost status, since forested areas reflected a deep permafrost table ( >2 m) associated with Pleistocene permafrost, while palsa-dominated landscapes were indicative of the presence of near-surface ( More