Ecology

Botanical content of the Libri Picturati Brazilian collectionOur identifications of all plant illustrations are listed with their vernacular names, page numbers, and associated information on growth form, geographical origin, conservation and domestication status in Supplementary Dataset S1. From the entire collection of Brazilian plant illustrations in the Libri Picturati, we identified 198 taxa that are organized in the Theatrum, LP and MC as indicated in Supplementary Table S1. Between folios 729 and 731 of the Theatrum, an illustration of a tea plant (Camellia sinensis (L.) Kuntze) is glued, which was sent by Cleyer from Batavia (currently Jakarta, Indonesia), the headquarters of the Dutch East Indian Company. As it was inserted later in the Theatrum and not depicted in Brazil, we did not include it in our analysis. A few plants remained unidentified due to a lack of morphological characters, the limited quality of the drawing and/or the lack of references to written sources by Marcgrave or Piso7,8.Among the LP botanical watercolors, we identified 34 vascular plant species (38 images) with the Passifloraceae as the most represented family (five species, six images), followed by the Fabaceae (five species, five images). Among the MC plant drawings, we identified 26 vascular plant species (34 images) and the most represented families were the Cucurbitaceae (three species, seven images) and the Myrtaceae (three species, three images). Among the illustrated content of the Theatrum, we identified 162 vascular plant species (175 images) and one basidiomycete fungus (Copelandia cyanescens (Sacc.) Singer, Bolbitiaceae). Fungi were commonly placed within the plant kingdom until the mid-twentieth century. The most represented families among the illustrated content are the Fabaceae (22 species, 22 images), followed by the Solanaceae (10 species, 11 images), Lamiaceae (six species, six images) and Myrtaceae (six species, eight images). The Fabaceae is the most diverse plant family in the world41, while the Myrtaceae is one of the most rich-species woody plant family in the Atlantic Forest in Brazil42.Mentzel’s unfinished task: the intended botanical content of the Theatrum
The Theatrum also includes 206 empty folios, interleaved between 160 folios with plant illustrations (see example in Fig. 1). On most folios, vernacular names and references to the pages of the HNB and IURNM are written on the top center, often relating to one taxon, but sometimes referring to two taxa (Fig. 1). This occurs specially at the end of the collection, as if the maker had ended up with little space and somehow had to squeeze them in. Among these unillustrated folios, the vernacular plant names and references to Marcgrave and Piso’s sources allowed us to identify 196 vascular plant species (218 records) including five ferns from the families Drypteriaceae (one species), Polypodiaceae (one species) and Pteridaceae (three species); one alga (Sargassum tenuissimum (Endlicher & Diesling) Grunow, Phaeophyceae) and a marine sponge (Clathria cf. nicoleae Vieira de Barros, Santos & Pinheiro, Microcioniadae) (Supplementary Dataset S1). Considering that the study of spongiology (Porifera) did not develop until the mid-nineteenth century, these animal colonies must have been considered an aquatic plant because of the tree-like shape and the fact of living attached to the seabed. The most represented family that would correspond to the empty folios was the Fabaceae (29 unillustrated species, 33 records), followed by the Arecaceae (nine species, ten records), Solanaceae (nine species, nine records), and Asteraceae (seven species, seven records). Estimates of the intended botanical content (i.e., empty folios with references together with the illustrated folios) are shown in Table 1.Figure 1Similar vernacular names for related taxa and distinct taxa associated to the same vernacular name in the Theatrum Rerum Naturalium.Full size imageTable 1 Estimations of the botanical content of the Theatrum Rerum Naturalium, including empty and illustrated folios.Full size tableOn p. 139 of the Theatrum, the vernacular name Ambaibuna is written on an empty page without reference to Marcgrave’s or Piso’s books. The page with Ambaibuna is located between Ambaiba (p. 137), which corresponds to the illustration of Cecropia pachystachya Trécul, and a blank page with only the vernacular name Ambaitinga (p. 141), which corresponds to C. hololeuca Miq.7: 92,24 (Fig. 1). The Brazilian Cecropia species are known in Tupi-related languages as Ambauba, Ambauva or Umbaúba (https://dataplamt.org.br/), which are phonetically and morphologically similar to Ambaibuna. For those reasons, we initially assumed that Ambaibuna referred to a Cecropia species, but the same name Ambaibuna is later repeated together with the name Iito (p. 227) next to an illustration that represents a completely different tree species: Guarea guidonia (L.) Sleumer (Fig. 1). Furthermore, the name Ambaibuna is also written above the illustration of a grapevine, Vitis vinifera L. (p. 257), also unrelated to Cecropia (Fig. 1).Whether Ambaibuna was a generic name to designate several non-related species or represents a mistake by the author who wrote the names on the illustrations remains unknown. On the other hand, neither Marcgrave nor Piso mentioned Ambaibuna in their descriptions of the Brazilian flora. Aside from Marcgrave and Piso’s books7,8, it is yet to be determined which source(s) Mentzel relied on when arranging the botanical content of the Theatrum. It is nonetheless clear that he must have been confused by the similarity of some of the Tupi-related plant names. Unfortunately, Marcgrave was no longer present to help him match the illustrations, names and descriptions, because he died about 16 years before Mentzel started organizing the Brazilian plant illustrations.Origin of the exotic species in the Libri Picturati
The Libri Picturati collection depicts in its majority native Brazilian plants. Most of the species represented in the Theatrum are native from Brazil, but the proportion of native species is much lower in the MC and lowest in the LP, in which almost half of the illustrations represent introduced species (Fig. 2).Figure 2Proportion of native and introduced species in the Brazilian collection of the Libri Picturati: Theatrum Rerum Naturalium (Theatrum), Libri Principis (LP) and Miscellanea Cleyeri (MC).Full size imageThere are 35 species of exotic origin in the complete Brazilian collection of the Libri Picturati (Supplementary Table S2). These introduced species now occur in (sub-) tropical areas around the world. Most of the exotic plants originally came from other parts of the Americas, especially Mexico, the Caribbean and the Andes region (14 species); followed by those that originated in the African continent (10 species) and tropical Asia (nine species) (Supplementary Table S2). Most of the exotic American plants that were introduced to Brazil were domesticated and traded by indigenous groups long before the European colonization, such as papaya (Carica papaya L.), cotton (Gossypium barbadense L.), sweet potato (Ipomoea batatas (L.) Lam.), beans (Phaseolus vulgaris L.), guava (Psidium guajava L.) and maize (Zea mays L.)37. Most of the species of Asiatic origin were already naturalized or cultivated in Africa and introduced to Brazil by means of the Trans-Atlantic slave trade before the Dutch arrived, such as yams (Dioscorea alata L.), plantains (Musa × paradisiaca L.) and weeds like Abrus precatorius L. and Plumbago zeylanica L.43,44. Others were introduced from Europe by merchants and settlers, such as the Portuguese Jesuits, who incorporated them as remedies into their boticas (Jesuit pharmacies in the colonies). For example, the various Citrus and pomegranate fruits were not only planted as fruits but also used to expel roundworms and to combat cold fevers, respectively45: 88. Before their arrival to Brazil, the Portuguese and Dutch must have been familiar with some African plants, such as Aloe vera (L.) Burm.f., Ricinus communis L. and Tamarindus indica L. These useful plants were already known in Europe through Arabic and Greek medical texts, which knowledge was boosted by their translations into Latin during the High Renaissance45,46. Punica granatum L. was introduced into the Iberian Peninsula via ancient merchant routes in the Mediterranean47 and brough to Brazil by the Portuguese45. Grapes (Vitis vinifera L.) were already cultivated by the Portuguese in Pernambuco around 154248. Along the Atlantic coast, lemons, pomegranates and grape vines adapted to the new environmental conditions and thrived in the vicinities of Johan Maurits’ residence, as evidenced by the illustrations in the Theatrum and textual accounts6,7,40.The presence of these globally commodified plants is common today in Brazil as in many regions worldwide. Other species seem to have lost their popularity over time. The so-called Ethiopian, Guinean or Negro pepper, Xylopia aethiopica (Dunal) A.Rich., was present around the 1640s in northeast Brazil, as evidenced in the Libri Picturati by a painting with a fruiting branch with leaves named Piperis aethiopici spés (Fig. 3a). The first iconography of this aromatic tree in Europe is found in Matthioli’s commentaries on Dioscorides under the name of Piper aethiopicum49: 575 and its fruits were previously cited by the Persian polymath Avicenna (980–1037)30. In Europe, this African pepper was commonly used until southeast Asian spices gained popularity in the sixteenth century50. In the plantation societies of tropical America, X. aethiopica constituted a food crop for enslaved Africans in the early colonial period43: 135. Today, its fruits are used in aphrodisiac tonics51 and special dishes prepared for African deities (Orishas) in Cuba43: 90, but it is unclear whether the species grows in Brazil. Its current distribution range encompasses West, Central and Southern Africa (https://gbif.org/occurrence/map?taxon_key=3157151). The dry fruits are used in tropical Africa as a condiment, in rituals and as medicine to treat cough, bronchitis, rheumatism, malaria, amenorrhea and uterine fibroids52,53,54. There is an herbarium record in Brazil made by photographer and anthropologist Pierre Verger. The label on the specimen mentions ‘Brazil’ and ‘Plantas de Candomblé’ and it indicates that the voucher was deposited at the Herbarium Alexandre Real Costa (ALCB, according to Index Herbariorum: http://sweetgum.nybg.org/science/ih/, accessed 23 August 2021) in Bahia (Verger s/n, ALCB012478, available at ALCB, via Species Link: https://specieslink.net/search/, accessed 23 August 2021) Verger presumably collected this specimen in Bahia in 1967 while he was researching on ritual and medicinal plants used in Candomblé (http://inct.florabrasil.net/alcb-resgate/, accessed 2 June 2021)55. However, it seems to be a mixed collection, as the leaves are oppositely arranged and with long petioles, which is uncommon to Annonaceae30. In Brazil, the fruits of the Brazilian relative Xylopia aromatica (Lam.) Mart. have probably served as a good substitute for X. aethiopica, as they have a similar peppery taste and stomachic properties56: 3, and are more easily gathered from the cerrado savannahs or the Amazon rainforest. Voeks57 documented X. aethiopica seed powder as used in Candomblé rituals by Yoruba practitioners in Bahia. Nevertheless, there is no clear information whether X. aethiopica is cultivated in the Neotropics or imported; thus, the origin of the fruits, seeds or its powder in Brazil remains uncertain.Figure 3 (a) The African spice-producing tree Xylopia aethiopica depicted in the Theatrum Rerum Naturalium (p. 321); (b) The first record of the sunflower (Helianthus annuus) in Brazil (Theatrum: 555).Full size imageThe first reference to the sunflower (Helianthus annuus L.) in Brazil dates to the twentieth century, when it was introduced by European immigrants due to its economic value as an oil-producing crop58. Sunflowers are of North American and/or Mexican origin 59,60, and were introduced to Europe in the sixteenth century by the Spanish, as part of the Columbian exchange61. Merchants observed how native Americans benefited from this plant and exported the sunflower to Europe, where it was primarily valued as ornamental and later as a food crop, propelled by genetic improvement by the Russians in the 1800s59. Before the sunflower became a popular and well-stablished crop in the twentieth century, this plant was already encountered in northeast Brazil, as evidenced by the illustration in the Theatrum (Fig. 3b). Portuguese sailors may have played a role in its introduction to Brazilian territories or it could have been intentionally brought by merchants or Jesuits, although the latter paid more attention to medicinal plants45,62. We may also consider the Dutch as active agents in its introduction to their colonies in the northeast. A relevant female agent in the dissemination of the sunflower in the Netherlands was Christine Bertolf (1525–1590), who was acquainted with the Spanish court and keen of the rare plants that thrived in the Royal Botanical Garden in Madrid63. She spread textual and visual information about the sunflower, and possibly also its seeds, among her network of naturalists and collectors, including the Flemish botanists Dodoens and Clusius63. After Dodoens64: 295 depicted the first European sunflower in his herbal in 1568, images and descriptions of this species began to circulate in manuscripts of other naturalists and physicians in Europe (e.g., Matthioli65: preface, Fragoso66: title page, Monardes67: 109 and Clusius68: 14–15). Thus, by the seventeenth century, Dutch scholars and collectors of exotic naturalia were familiar with sunflowers, which possibly promoted its cultivation at Johan Maurits’ gardens with ornamental purposes.Interestingly, the sunflower is referenced as Camará-guaçú, an indigenous term from the macrolinguistic Tupi family. Camará, Kamará or Cambará is a generic name given to several unrelated species, such as Lantana camara L. (Verbenaceae) and Ageratum conyzoides L. (Asteraceae) (http://www.dataplamt.org.br/, accessed 2 June 2021), both found in the Theatrum (p. 341 and 343 respectively). According to Tibiriçá69, in Tupi caa means plant and mbaraá means illness, and according to Cherini70 Cambará means “leaf of rough bark”. Hence, Camara also refers to medicinal plants with rough leaves in general. Guaçú means big and miri small71, which matches with the larger inflorescence of H. annuus in contrast to the African weed Sida rhombifolia L. (Malvaceae), documented as Camara-miri in the HNB and “used by black people as a broom to sweep the houses of their masters”7: 110. According to the Tupi-based nomenclature associated to H. annuus in the Theatrum, Tupi indigenous groups were already familiar with the sunflower in Brazil around the 1640 s.Life forms and domestication status of the Libri Picturati plantsMost of the species in the Theatrum are tropical trees, followed by shrubs, herbs, and lianas (Fig. 4). Several are rainforest trees, such as Andira fraxinifolia Benth., Garcinia brasiliensis Mart. and Syagrus coronata (Mart.) Becc. The same trend was observed for the illustrations in the MC, with trees as the most often represented life forms, followed by shrubs, lianas and herbs. Typically, the LP contains much less trees, but more small herbs, shrubs and vines that were probably found in and around Mauritsstad (i.e., the former capital of Dutch Brazil, currently a part of the Brazilian city of Recife), such as Commelina erecta L. and Turnera subulata Sm., which commonly grow in disturbed landscapes.Figure 4Proportion of life forms of the species depicted in the Brazilian collection of the Libri Picturati: Theatrum Rerum Naturalium (Theatrum), Libri Principis (LP) and Miscellanea Cleyeri (MC).Full size imageAlthough the majority of the species depicted in the Theatrum and the MC are wild forest trees, some species are found both in the wild and cultivated, such as Psidium guineense Sw., which was part of the pre-Columbian anthropogenic forests or ‘indigenous landscape’ in Brazil37,38,72. Some trees were planted in or around Recife. Hancornia speciosa Gomes, known by its Tupi-based name Mangabiba or Mangaiba [Mangabeira]7: 121, was cultivated in Mauritsstad6: 242,40. The fruit of H. speciosa (Mangaba) was harvested in great amounts as it was a highly appreciated food7: 122. Seeds were collected to plant the tree, and Marcgrave gave details about the specific locations of varieties in different northeastern locations (Salvador, Sergipe and Olinda). H. speciosa was already selected and managed by indigenous groups before colonization37, yet wild populations of this tree are still found in the Brazilian rainforest and savannah (http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB15558, accessed 4 June 2021).Domesticated plants are represented in higher proportions within the LP and the MC (Fig. 5), accounting mostly for introduced fruit species (Supplementary Dataset S1), such as Citrus spp., Musa x paradisiaca and Cocos nucifera L., which were cultivated in Maurits’ gardens in Recife40. The influence of the European colonization of Brazil is also visible by the presence of weeds from Asia and Africa among the illustrations in the Theatrum and the LP, such as Abrus precatorius L., Argemone mexicana L., Boerhavia coccinea Mill. and Plumbago zeylanica L. Some of these plants were introduced from Africa via the slave ships, while others may have dispersed naturally44. Guilandina bonduc L., an African scrambling shrub depicted as Inimboi in the Theatrum, was described by Piso7: 95 as “growing in abundance in sandy and dry forests of the coasts”. We categorized G. bonduc as a wild plant: its round seeds could have been brought by oceanic currents from West African shores and germinated in the coastal vegetation of Pernambuco and other South American areas73. However, G. bonduc may also have reached Brazil during the Trans-Atlantic slave trade, as the hard, grey seeds are used in the African game Oware and also used in bead ornaments74.Figure 5Domestication status of the species in the Brazilian collection of the Libri Picturati: Theatrum Rerum Naturalium (Theatrum), Libri Principis (LP) and Miscellanea Cleyeri (MC).Full size imagePlant parts represented in the Libri Picturati
The way plants are depicted in the Libri Picturati provides us with information about the level of botanical skills of the artists, and how closely they worked together with the naturalists in the Dutch colony. Some plants are represented by only loose parts or depicted sterile, while others show us different organs and reproductive stages, which greatly facilitated their taxonomic identification (Table 2).Table 2 Plant parts represented in the botanical illustrations of the Libri Picturati: Theatrum Rerum Naturalium (Theatrum), Libri Principis (LP) and Miscellanea Cleyeri (MC).Full size tableMost illustrations depict fertile plant species with flowers and fruits, often cut in half to show the seeds, which reveals a high level of botanical knowledge. Fertile plants are more common in the Theatrum, in a few occasions also showing their tubers, such as Spondias tuberosa Arruda, known as Umbi [Iva Umbu], of which the prominent tuber in the bottom front captures the attention of the observer (Fig. 6a). Likely associated to a scientific purpose, drawing some plant parts out of proportion corresponds to a pictorial style also observed in other iconographies. This is also the case in the Icones Plantarum Malabaricarum, which depicts plants from Ceylon (modern Sri Lanka) in the eighteenth century and often accentuates useful fruits, flowers or roots75.Figure 6 (a) Spondias tuberosa with the tuber painted in front of the branch with leaves, tiny white flowers and a detail of the immature (green) and mature (yellow) fruit in the back (Theatrum Rerum Naturalium: 261); (b) Infertile individual of Hippeastrum psittacinum (Theatrum: 389); c Ficus gomelleira leaf, probably picked from the ground (Theatrum: 157); (d) Flowering vine of Centrosema brasilianum (Libri Principis: 1); (e) Amphilophium crucigerum dry open fruit without seeds (Theatrum: 387).Full size imagePiso7: 78 indicated that roots [tubers] of S. tuberosa deserved special attention, because of the way they developed underground and their use as a refreshment [water reservoir] for feverish patients and exhausted travelers, as he experimented himself. He and Marcgrave7: 108 also described how its fruits were valued as food. This example not only provides textual and visual evidence of the field trips to the interior by these naturalists and their first-hand experiments, but also adds insights into the connectedness between artistic and scientific practices in seventeenth century Dutch Brazil. Currently S. tuberosa, known as Umbu or Umbuzeiro (https://dataplamt.org.br/), is an important economic and subsistence food resource for rural communities in semiarid regions of northeast Brazil76,77. Its specialized root system (xylopodia) bears tubers that store liquids, sugars and other nutrients and allow the survival of the tree during the dry seasons of the caatinga and central Brazilian savanna, where this species is endemic78. The water or sweet juice of these xylopodia is still used as an emergency thirst quencher in extreme arid areas of the Brazilian sertão79; also see https://www.youtube.com/watch?v=NyGNlrljAww, accessed 25 August 2012].In the Theatrum, a small proportion of plants are illustrated in their sterile stage, such as Hippeastrum psittacinum (Ker Gawl.) Herb. (Fig. 6b) or Ficus gomelleira Kunth & C.D.Bouché (Fig. 6c). Marcgrave7: 32 did not see the impressive flower of H. psittacinum as it is lacking in his observations25: 59. The Theatrum painting must have been made in the wet season in the interior of Pernambuco, when Marcgrave and the painter(s) encountered the lily with only leaves, before these fall off and make place for the mesmerizing flower25. Ficus gomelleira, depicted by a single oblong leaf with its characteristic pinnate venation (p. 157), is a large tree, up to 40 m tall (https://portal.cybertaxonomy.org/flora-guianas/node/3041, accessed 4 June 2021). It can be challenging to collect a branch, so the painter(s) or local assistants possibly picked a leaf from the ground (Fig. 6c).The LP contains mainly flowering plants (e.g., Ruellia cf. elegans Poir.), tendrillate vines (e.g., Centrosema brasilianum (L.) Benth. (Fig. 6d)) and cultivated crops, such as peanuts (Arachis hypogaea L.), pumpkins (Cucurbita pepo L.) or guava (Psidium guajava L.) (Supplementary Dataset S1). Compared to the MC and the LP, a smaller proportion of the illustrations display only flowers or fruits in the Theatrum. Yet, these deserve special attention as the reasons for only painting the reproductive organs in the three collections may differ. While in the MC and LP flowers or fruits represent species that are domesticated or more likely to be found in urban areas, such as Capsicum baccatum L. or Hancornia speciosa, the Theatrum contains more loose parts of native plants found in the rainforest. Amphilophium crucigerum (L.) L.G.Lohmann is a liana referenced by the Tupi-related name Iaruparicuraba (Theatrum: 387) and today known in Brazil as pente-de-macaco (https://dataplamt.org.br/) due to its large dehiscent fruit (c.17 cm long) that opens in two valves covered with soft spines, hence its name “monkey’s comb” (Fig. 6e). Its winged seeds are not present in the drawing, possibly one empty valve was gathered from the ground, and the seeds were already dispersed by the wind.In the MC, we also find some drawings of infertile structures, but these mostly belonged to species that were depicted on several folios. When assembling those folios, we observed the whole plant represented in its fertile stage: the watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is depicted with its leaves and fruit on folio 63 (verso) and its leaves on folio 64 (recto). In the case of Furcraea foetida (L.) Haw., whoever bounded the drawings in the MC collection did not realize that folios 63 (recto), 64 (verso) and double folio 68 formed together one entire plant (See Supplementary Fig. S1).On other occasions, the painters focused on painting the plant parts that were valuable to humans. Several rainforest trees were highly valued for its edible fruits or seeds, such as Hymenaea courbaril L.7: 101 or Lecythis pisonis L., of which the “seeds (also called chestnuts) were eaten raw or roasted”7: 128 and “were considered aphrodisiacs”7: 65. The fruit of Macoubea guianensis Aubl. was “appreciated for its sweetness by the indigenous peoples to eat during their travels, while Europeans used it to treat chest affections”8: 242. The fruit of Swartzia pickelii Killip ex Ducke was “not eaten unless it was cooked, from which the inhabitants made a wholesome delicacy for the stomach called Manipoy”8: 165. The same applies to the tomato-like fruits of the African eggplant Solanum aethiopicum L., which were “eaten cooked, after seasoning with oil and pepper; it has lemon taste”7: 24. While these plants are represented in the Theatrum only by their fruits (Supplementary Dataset S1), the tree branches or the whole plant are depicted in the written sources. The illustrations in the books were most likely made by Marcgrave, who aimed to describe and depict as many plant parts as possible, although compromising in aesthetic aspect. The painters, on the other hand, focused on the edible parts without sacrificing their aesthetics. In any case, the illustrations from the Theatrum and the woodcuts and descriptions in the HNB and IURNM often complemented each other and thus facilitated our identifications.Current conservation status of the Brazilian species in the Libri Picturati
In the past centuries, the Atlantic Forest and savannah regions of northeast Brazil have been severely affected by habitat loss and degradation due to the expansion of urbanization, intensive agriculture, farming and logging80,81. Several plant species that were abundant enough to be noted by European artists around 1640 are not common anymore today. According to the IUCN Red List, eight species in the Libri Picturati, seven in the Theatrum and one in the LP are currently experiencing population decline or are at risk of facing extinction (Supplementary Table S3). Several endemic plants from the northeast Atlantic rainforest and caatinga biomes appear in the illustrations. Four species in the Libri Picturati are currently CITES-listed and restricted to trade: the cacti Brasiliopuntia brasiliensis (Willd.) A.Berger, Cereus fernambucensis Lem., Epiphyllum phyllanthus (L.) Haw. and Melocactus violaceus subsp. margaritaceus N.P.Taylor The latter is an endemic cactus of the coastal sand dunes’ ecoregion in the Atlantic rainforest known as restinga, which is severely threatened by agricultural expansion and urbanization82.Some endemic species are classified as Least Concern by the IUCN or the CNC Flora (12 species), while others (13 species) have not been evaluated yet (Supplementary Dataset S1). The MC does not contain threatened species, but includes two endemic trees: Attalea compta Mart. and Eugenia cf. brasiliensis Lam., which are only found in the biodiversity hotspots of the Atlantic rainforest and the cerrado, both greatly affected by habitat loss23. The mangrove vegetation along the Brazilian coast has been severely affected by urbanization, pollution by industrial and domestic waste and climate change83,84, threatening the populations of the mangrove trees Avicennia schaueriana Stapf & Leechm. ex Moldenke and Laguncularia racemosa (L.) C.F.Gaertn. The occurrence of anthropogenic impacts and the lack of available data call for the implementation of more in-depth and continuous studies on the conservation status of these vulnerable populations.Linking the plant illustrations to the published works and Marcgrave’s herbariumA total of 357 different plant species is described in the HNB and IURNM (Supplementary Dataset S2). Because the Theatrum constitutes a larger number of illustrations, we found more taxa from the books and the herbarium represented in this source (102 out of 163, 63%). However, the largest overlap was found between the MC and the HNB / IURNM: 21 out of 26 taxa (81%) were also described in the books. A smaller overlap exists between the LP and the HNB / IURNM (18 out of 34, 53%). We counted 143 taxa at species level in Marcgrave’s herbarium (Supplementary Dataset S3) and we observed some of these preserved species in all three pictorial works, with the largest percentage of taxa in common with the MC (seven out of 26, 27%), probably because of its smaller number of images. Strikingly, a third of the species illustrated in the whole Brazilian collection of the Libri Picturati could not be ascribed to the species described by Marcgrave or Piso (61 out of 180, 34%). More

Acinetobacter isolates from the TGR regionIn the TGR region, we isolated 21 Acinetobacter strains (one A. johnsonii, one A. haemolyticus and 19 Acinetobacter sp. strains) (Table S1). Based on phylogenetic analysis after 16S rRNA gene sequencing, these 21 isolates belong to the genus Acinetobacter, exhibiting a similarity of 95.38–99.93% with known Acinetobacter strains in GenBank (Table S1). In phylogenetic tree (N-J) constructed with both isolated and known Acinetobacter strains, these 21 isolates branched deeply with three Acinetobacter clusters consisting of important clinical Acinetobacter species, such as A. johnsonii H10 (FJ009371), A. junii NH88-14 (FJ447529), A. baumannii ATCC19606T (HE651907), A. lwoffii DSM2403T (X81665) and A. haemolyticus TTH04-1 (KF704077) (Fig. 1). Five reference Acinetobacter strains were selected and used21. Currently, the genus Acinetobacter comprises 68 species with validly-published names (https://apps.szu.cz/anemec/Classification.pdf, May 25, 2021). Among the named species, A. baumannii is the most studied species associated with clinical infections followed by the non-A. baumannii species A. haemolyticus, A. junii, A. johnsonii, and A. lwofii21.Figure 1A phylogenetic tree of 16S rRNA gene sequences showing position of isolates among species of genus Acinetobacter. Both isolates from the TGR region (the bold fonts) and reference strains used to infect Caenorhabditis elegans (the red fonts) are shown.Full size imageEffect of different Acinetobacter strains isolated from the TGR region and reference strains on lifespan of nematodesL4-larvae were exposed to different Acinetobacter strains for 24-h. Totally 21 Acinetobacter strains isolated from the TGR region and 5 reference strains of Acinetobacter species were used for the lifespan analysis. Based on the comparison of lifespan curves, exposure to Acinetobacter strains of AC2, AC3, AC4, AC5, AC6, AC7, AC8, AC9, AC10, AC11, AC12, AC13, AC14, AC16, AC17, AC19, AC20, A. johnsonii H10, and A. haemolyticus TTH0-4 could not alter lifespan curve (Fig. 2). Similarly, Acinetobacter strains of AC2, AC3, AC4, AC5, AC6, AC7, AC8, AC9, AC10, AC11, AC12, AC13, AC14, AC16, AC17, AC19, AC20, A. johnsonii, and A. haemolyticus also could not influence mean lifespan (Fig. 2). Different from these, the lifespan curves of nematodes exposed to Acinetobacter strains of AC1, AC15, AC18, AC21, A. baumannii ATCC 19606T, A. junii NH88-14, and A. lwoffii DSM 2403T were significantly (P  More

Batrachochytrium salamandrivorans (B. salamandrivorans) culture conditions and zoospore isolationB. salamandrivorans type strain (AMFP 13/01)8 was grown in tryptone-gelatin hydrolysate-lactose (TGhL) broth and incubated for 5−7 days at 15 °C. Zoospores were harvested by replacing the TGhL broth with distilled water. The collected water was filtered through a sterile mesh filter with pore size 10 µm (Pluristrainer, PluriSelect) to remove sporangia. Zoospore viability and mobility were confirmed using light microscopy.Salamander skin lysate binding assayBinding of B. salamandrivorans spores to the protein or carbohydrate fractions from fire salamander (Salamandra salamandra) skin was tested by treating fire salamander sloughed skin lysates enzymatically with glycoside hydrolases, followed by protein precipitation. An overview of the skin lysate binding assay is shown in Supplementary Fig. 3.To collect the sloughed skin, ten captive-bred adult fire salamanders were housed at 15 ± 1 °C on moist tissue. The sloughed skin samples were ground with liquid nitrogen into a fine powder and then homogenized, using 3 ml RadioImmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) per gram of tissue. Samples were incubated for 1 h at 4 °C, centrifuged at 27.000 × g for 10 min and the supernatant was subsequently collected. Protein concentration was determined using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). The obtained skin lysate was equally divided, one part was treated with Protein Deglycosylation Mix II and two parts were kept as crude skin lysates. Protein Deglycosylation Mix II (New England BioLabs) was used to remove N-linked and O-linked glycans from glycoproteins. According to the manufacturer’s instructions, 5 µl 10× Deglycosylation Mix Buffer I and 5 µl Protein Deglycosylation Mix II were added to 40 µl skin lysate. The mixture was incubated at 37 °C for 16 h. Protein precipitation was conducted on the redundant Protein Deglycosylation Mix II treated and crude skin lysates. The precipitation was performed by slowly adding saturated ammonium sulfate solution to the skin lysates to achieve a final concentration of 75%. Samples were then centrifuged at 21.130 × g for 30 min to separate the precipitated proteins from the supernatant. The precipitated protein pellets were resuspended in 300 µl of 0.05 M carbonate−bicarbonate coating buffer (3.7 g NaHCO3, 0.64 g Na2CO3, 1 L distilled water, pH 9.6). Each skin lysate solution was adjusted to the volume of 300 µl by adding a coating buffer. One hundred µl of each skin lysate solution was coated in each well of 96-well polystyrene microtiter plates (MaxiSorpTM plate, Thermo Fisher Scientific) in three technical replicates. As controls, coating buffer (negative control) and 75% ammonium sulfate solution were also coated on the 96-well plates. After incubation at 4 °C for 24 h the coated plates were washed three times with washing buffer (0.01 M PBS-Tween 20, pH 7.4) and blocked with 1% BSA overnight at 4 °C. Plates were then again washed three times with washing buffer and three times with distilled water. One hundred µl of B. salamandrivorans zoospore suspension (1 × 107 zoospores per ml) were added in each well. Plates were incubated for 20 min at 15 °C and washed five times with distilled water to remove the unbound zoospores. Digital photographs were taken through via an inverted light microscope at 100 × magnification. Five pictures were taken for each well and zoospores in each photograph were counted in a blind fashion. Three independent repeats of the experiment were conducted (biological replicates).Carbohydrate binding assayTo further determine which carbohydrates expressed on fire salamander sloughed skin can mediate the binding of B. salamandrivorans zoospores, B. salamandrivorans binding against four carbohydrates; N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), mannose, and lactose was tested. The three monosaccharides and the disaccharide (Sigma-Aldrich) were dissolved and thereafter diluted in coating buffer to achieve a concentration of 5% (w/v). Then they were coated in triplicate wells by incubating at 4 °C for 24 h42. Plates were rinsed three times with washing buffer and blocked with 1% BSA overnight at 4 °C.Hundred μl of B. salamandrivorans zoospore suspension (1 × 107 zoospores per ml) was added in each well and incubated for 20 min at 15 °C. After washing the wells five times with distilled water to remove unbound zoospores, the plates were evaluated using a light microscope. Digital photographs were taken at 100 × magnification. Five pictures were taken for each well and zoospores in each photograph were counted in a blind fashion. Three independent repeats of the experiment were conducted (biological replicates).In this experiment the highest level of B. salamandrivorans spores binding to lactose was observed. Lactose is a dissacharide consisting of glucose and galactose. Therefore, in the following experiments galactose, glucose and their derivatives will be tested separately.Carbohydrate chemotaxis testChemotaxis of B. salamandrivorans toward free carbohydrates was tested as previously explained (Supplementary Fig. 4)12. The sugars D-Glucose (Sigma-Aldrich), D-mannose (Sigma-Aldrich), Lactose (Sigma-Aldrich), and D-galactose (Sigma-Aldrich) were tested as attractant for B. salamandrivorans. The monosaccharides instead of the amide derivatives were used in this experiment to exclude any chemotactic signalling activity of the amides. Sugars were dissolved in distilled water, filter sterilized, and tested at a 0.1 M concentration. Hematocrit capillaries (75 mm length; Hirschmann laborgeräte, Eberstadt, Germany) were filled with 60 µl carbohydrate solution, vehicle control capillaries with 60 µl sterile distilled water. To prevent leakage, the capillaries were sealed with wax plugs (Hirschmann laborgeräte, Eberstadt, Germany) at one side. Each capillary was swiped on the outside with lens paper (Kimtech Science, Kimberley Clark, Roswell, GA, USA) to remove possible attractant spillover. Capillaries were incubated in 400 µl inoculum containing 106 B. salamandrivorans zoospores in water and placed in a holder inclined about 65° upwards. The assay was incubated for 90 min at 15 °C, after which the capillaries were removed and swiped again at the outside to remove B. salamandrivorans zoospores possibly adhering on the outside. Inocula were checked for motility of the zoospores using an inverted microscope (Olympus CKX 41, Hamburg, Germany). Contents of the capillaries were collected and centrifuged for 2 min at 16.000 × g. The supernatant was removed as much as possible. The pellet was suspended in 100 µl Prepman Ultra Sample Preparation reagent (Applied Biosystems, Life Technologies Europe, Ghent, Belgium) and DNA was extracted according to the manufacturer’s guidelines. For each sample, the number of B. salamandrivorans zoospores was quantified using quantitative real-time PCR (qPCR)41, and data were analyzed using the Bio-Rad CFX manager 3.1. The primers and probe can be found in Supplementary Table 11. Within each assay, all carbohydrates and negative controls were tested at least in triplicate (technical replicates) and three independent repeats of the assay were performed (biological replicates).Carbohydrate transcriptome testRNA preparation: total RNA was isolated from B. salamandrivorans zoospores treated with different carbohydrates. Therefore, newly released zoospores (less than 2 h after induction of spore release by adding water) were harvested from 175 cm2 cell culture flasks by replacing the TGhL broth with distilled water, which was filtered using a sterile mesh filter with pore size 10 µm (Pluristrainer, PluriSelect). Six-biological replicates containing 4 × 107 zoospores were obtained. Each biological replicate consisted of a pool of spores harvested from three cell culture flasks. Per biological replicate, the spores were divided into 4 eppendorfs (107 zoospores/eppendorf) which were treated for 1 h at 15 °C with H2O (control), 50 mM (D-galactose), 50 mM (D-glucose), or 50 mM (D-mannose) (Supplementary Fig. 5). After 1 h, the zoospores were centrifuged for 5 min at 4.000 × g at 15 °C to remove the supernatant, after which RNA was extracted using the RNeasy mini kit (Qiagen)18. The RNA was treated with Turbo™ DNase (Ambion), following the manufacturer’s instructions. RNA degradation and contamination were monitored on 1% agarose gels. The RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). Finally, the RNA integrity and quantitation were assessed using the RNA Nano 6000 assay kit of the Bioanalyzer 2100 system (Agilent Technologie, CA, USA).Library preparation for transcriptome sequencing: Whole-transcriptome sequencing libraries were constructed and sequenced on the Illumina HiSeq platform (Novogen, China). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150−200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.Clustering and sequencing: The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.Quality analysis, mapping, and assembly: Raw data (raw reads) of FASTQ format were first processed through fastp (version 0.20.0). In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. At the same time, Q20, Q30, and GC content of the clean data were calculated (Supplementary Table 12). All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly. Paired-end clean reads were mapped to the B. salamandrivorans reference genome using HISAT2 (version 2.0.5) software18. Featurecounts (version 1.5.0-p3) were used to count the read numbers mapped to each gene, including known and novel genes (Supplementary Table 13). And then RPKM (reads per kilobase per million) of each gene was calculated based on the length of the gene and reads count mapped to this gene.Gene expression, differential expression, enrichment, and coexpression- analysis: Differential expression analysis was performed using the DESeq2 R package43. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). Genes with an adjusted P-value < 0.05 found by DESeq2 were assigned as differentially expressed. Protein domains were annotated with PFAM version 27 and 33 and KEGG domains, Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package44 and dcGOR R package45. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. ClusterProfiler R package44 was also used to test the statistical enrichment of differentially expressed genes in KEGG pathways.Detection of protease activityThe influence of carboydrate exposure on protease activity of B. salamandrivorans zoospores was assessed. Therefore, zoospores were harvested from 175 cm2 cell culture flasks by replacing the TGhL broth with distilled water, which was filtered using a sterile mesh filter with pore size 10 µm (Pluristrainer, PluriSelect). A pool containing approximately 5 × 107 zoospores/ ml was obtained. 200 µl of the spore suspension (107 spores) was added to eppendorfs containing 200 µl H2O (H2O; n = 3), 200 µl 100 mM D-Glucose (Glc; n = 3), 200 µl 100 mM D-mannose (Man; n = 3), 200 µl 100 mM D-galactose (Gal; n = 3), or as a control, 200 µl H2O containing protease inhibitor mix (P8215, Sigma-Aldrich) (PI; n = 3). After 1.5 h at 15 °C, the zoospores were centrifuged for 5 min at 4.000 × g at 15 °C and the supernatant was collected. Protease activity in the supernatant was analyzed using the Pierce Fluorescent Protease Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Three independent repeats of the experiment were performed (biological replicates).Identification of B. salamandrivorans lectin genesPotential candidates of carbohydrate-binding molecules (CBMs) were identified in the B. salamandrivorans (AMFP) genome listed in the NCBI database (Bioproject PRJNA311566).B. salamandrivorans (AMFP 13/01) coding regions from the single annotated genome present on NCBI database (Bioproject PRJNA311566) were used to single out potential lectin genes of interest that could serve as genes of carbohydrate-binding proteins. The lectin candidates were identified with BLASTp (BLAST + 2.9.0) over the FungiDB database (constituting 199 candidates, database accessed 1st March 2018) using the stringent e-value cutoff of 1e−50 to avoid spurious hits46,47.From these, five candidates that referred to lectins and carbohydrate-binding were manually selected using the NCBI CDD (v3.16) conserved domain software with default settings48.Expression of two of these genes (BSLG_00833 and BSLG_02674) was confirmed by a previous mRNA expression analysis (Bioproject PRJNA311566)18.AnimalsThe animal experiments were performed following the European law and with the approval of the ethical committee of the Faculty of Veterinary Medicine (Ghent University EC) (EC2015/86). Only captive bred animals were used. Fire salamander larvae belonging to different life stages49 were used in a B. salamandrivorans infection trial.For lectin-histochemical staining, skin samples were collected from amphibian species Salamandra salamandra (n = 10), Ichthyosaura alpestris (n = 12), Lissotriton helveticus (n = 13), Pleurodeles waltl (n = 11), Lissotriton boscai (n = 3), Alytes obstetricans (n = 10), Cynops pyrrhogaster (n = 3), Triturus anatolicus (n = 3), Triturus marmoratus (n = 3), Calotriton asper (n = 10), Bombina variegata (n = 5), Rana temporaria (n = 10), Epidalea calamita (n = 5), Pelobates fuscus (n = 5) and Salamandra lanzai (n = 3). Tail or toe clips, ventral and dorsal skin samples were collected from animals that were euthanized with natrium pentobarbital 20% (KELA). The collected samples were immediately fixed in Bouin’s solution for 24 h.Mucosome samples were collected by bathing animals in HPLC-grade water for 1 h from 21 amphibian species (different animals as the ones used for the tissueclips), namely Lissotriton helveticus (n = 3), Pleurodeles waltl (n = 3), Lissotriton boscai (n = 3), Triturus anatolicus (n = 3), Triturus marmoratus (n = 3), Cynops pyrrhogaster (n = 3), Ichthyosaura alpestris (n = 3), Salamandra salamandra (n = 3), Lyciasalamandera helverseni (n = 3), Speleomantes strinatii (n = 2), Paramesotriton hongkongensis (n = 2), Plethodon glutinosus (n = 2), Chioglossa lusitanica (n = 3), Pachyhynobius shangchengensis (n = 3), Calotriton asper (n = 3), Salamandra algira (n = 3), Salamandra lanzai (n = 2), Alytes obstetricans (n = 3), Bombina variegata (n = 2), Epidalea calamita (n = 3) and Pelobates fuscus (n = 3).Exposure of fire salamander larvae and metamorphs to B. salamandrivorans Twenty-two early-stage and 26 late-stage larvae49,50 were inoculated with 1.5 × 105 B. salamandrivorans spores per ml water during 24 h. Ten days after the inoculation all the early-stage and sixteen late-stage larvae were euthanized. The two hind legs were analyzed by qPCR to detect the B. salamandrivorans GE load. A tail clip was stained with fluorescein-labelled RCA I (see below). Ten late-stage larvae were further kept until five weeks after metamorphosis.Six one-week-old fire salamander metamorphs were inoculated with 1 ml of water containing 1.5 × 105 spores for 24 h. The animals were euthanized 10 days after inoculation. The two hind legs were analyzed by qPCR to detect the B. salamandrivorans GE load. A tail clip was stained with fluorescein labelled RCA I (see below).Lectin-histochemical stainingFluorescein labelled RCA I (Ricinus communis agglutinin I) (Vector Laboratories) and Con A (Concanavalin A) has been used to detect the expression of galactose and mannose or glucose in the epidermis of amphibians38.After 24 h fixation in Bouin’s medium (Sigma-Aldrich), samples were washed first with tap water until the water ran colourless, then washed for 24 h in 70% ethanol saturated with lithium carbonate (Sigma-Aldrich) to remove picric acid. Tissues were then dehydrated in a graded ethanol series, cleared in xylene, embedded in paraffin, and sectioned in 4−6 µm slices. Before lectin staining, the sections were deparaffinized in xylene and hydrated in a series of ethyl alcohols. For better presenting the carbohydrate antigens, we performed antigen retrieval by submerging slides in citrate buffer (10 mM citric acid, pH 6.0) and heat treating in microwave (850 W for 3.5 min plus 450 W for 10 min). The slides were rinsed with PBS (0.01 M, pH 7.4) and immersed in 1% BSA (Sigma-Aldrich) for 15 min, to prevent non-specific lectin binding. Subsequently, the sections were incubated with either lectin RCA I (15 µg/ml) or lectin Con A (5 µg/ml) for 30 min. Lectins were diluted with lectin binding buffer (10 mM Hepes, 0.15 M NaCl, pH 7.5). As a negative control, lectin RCA I was mixed with 200 mM galactose, and lectin Con A was mixed with 200 mM mannose + 200 mM glucose, before incubating with skin sections to inhibit lectin binding. For positive control, a slide of fire salamander ventral skin sample for RCA staining, and midewife toad ventral skin sample for Con A staining, was included in each experiment. The slides were then washed in PBS, and cell nuclei were stained with 10 µg/ml Hoechst 33342 Solution (Invitrogen). Coverslips were mounted with ProlongTM Gold Antifade Reagent (Invitrogen). Staining results were observed using a Leica fluorescence microscope under 10× magnification, with a 450−490 nm BP excitation filter for lectin staining and a 355−425 nm BP excitation filter for Hoechst staining. Staining pictures were taken using Leica Application Suite (LAS) X software. The lectin staining intensities were classified as intense (3), strong (2), weak (1), or negative (0) staining (Supplementary Fig. 6). Experimental positive and negative controls were defined as intense (3) and negative (0) stained, respectively, and other slides were then evaluated in comparison to the set parameters. Hoechst staining results were paired with corresponding lectin staining results, making it easier to discern the tissue structure from the dark background. The fluorescent intensities were scored by three reviewers, respectively scoring the same dataset of pictures blinded three separate times, and the mean value was taken as the final result.Free galactose, mannose, and total carbohydrates in amphibian mucosomeMucus was collected from 21 amphibian species (see above). The animal body surface and volume of bathing water were calculated as follows: surface area of anuran species in cm2 = 9.9* (mass in g)^(0.56), surface area of urodelan species in cm2 = 8.42* (mass in g)^(0.694), and the quantity of HPLC-grade water to add to both anuran and urodelan species was determined by dividing the surface area by 4), and animals were bathed in respective amounts of HPLC-grade water for 1 h40,51. Animal washes were collected and concentrated by SpeedVac Vacuum Concentrators (Thermo Fisher Scientific) to 100 µl. The quantities of free galactose, mannose, and total carbohydrates in 100 µl of concentrated animal wash were measured using the Galactose Assay Kit (Abcam), Mannose ELISA Kit (Aviva Systems Biology), and Total Carbohydrates Assay Kit (Abcam), as per instructions. Concentrations of free galactose, mannose, and total carbohydrates in animal washes were divided by animal body surface to get the final results of sugar concentrations per square centimetre of the body surface.Statistical analysisStatistical analyses of fire salamander skin lysate binding assay, carbohydrate-binding assay, chemotaxis assay, and protease activity assay were performed using R version 4.0.3. To account for the experimental design, Generalized Linear Mixed Models (GLMM, R library lme452) were used, specifying a nested random effect whereby technical replicates are nested within biological replicates. Count data were modelled first using a Poisson distribution, but as significant overdispersion was present in the data, a negative binomial error structure was implemented. For the protease activity assay, data do not represent counts and a log transformation on the raw values were used to ensure normality of model residuals (Shapiro-Wilk W  > 0.95) allowing a Gaussian error structure (i.e., a Linear Mixed Model (LMM). To test for differences between categories, the (G)LMMs were directly fed to the glht function of the R library multcomp53, setting up contrasts for Tukey’s all-pair comparisons, resulting in Bonferroni-corrected p-values adjusted for multiple testing. Statistical analyses of the larvae infection trial were performed in R version 4.0.0, with tidyverse54 version 1.3.0, MASS55 version 7.3-51.6, VGAM56 version 1.1-3, DHARMa57 version 0.3.1 and glmmTMB58 version 1.0.2.1. Infection loads of larvae and metamorphs were compared, using the Wilcoxon rank sum test, formula Chytrid GE load ~ larvae vs metamorph status, from the stats package. The correlation between larvae Ricinus communis agglutinin (RCA) scoring (1 = weak staining, 2 = strong staining, 3 = intense staining) and infection load was performed using the glm() function on log-transformed genomic equivalents with formula log10(B. salamandrivorans load in Genomic equivalents)~ RCA score, treating RCA score as an ordered factor with guassian distribution. As non-transformed chytrid loads showed zero-inflation and overdispersion, we also fit a generalized linear model with negative binomial distribution (GE load ~ RCA score) with RCA score as an ordered factor, using glmmTMB with a zero-inflation model (~ RCA score), which showed a comparable positive correlation between RCA score and GE load (conditional model coefficient = 5.67, p = 0.003, zero-inflation model coefficient = −2.21, p = 0.016). Residuals and chi-square test indicated the negative binomial model was not a significant improvement and so the simpler generalized linear model on transformed data was included. RCA scoring and larval stage prediction probabilities in Fig. 4c were generated by polr(RCA score ~ life stage) from MASS. Model fit and appropriateness was tested using Chisq test (p = 0.003), the model fit compared favourably to a more complex multinomial logit model and a model fit based on 70% of the data predicted 70−75% of remaining data (when data repeatedly sampled with different seeds, with the final model fit to all data).The regression and correlation analyses of different amphibian species were performed in SPSS (IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY, USA). Correlations of RCA scores with B. salamandrivorans infection peak loads, mortality rates, and percentage of free galactose were calculated by two-tailed Point-Biserial Correlation (p  More

WHERE I WORK
04 October 2021

The Māori meeting house that’s also a research lab

Ocean Mercier researches how Indigenous knowledge and Western science can help resolve environmental issues.

James Mitchell Crow

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James Mitchell Crow

James Mitchell Crow is a freelance writer in Melbourne, Australia.

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Ocean Mercier is an associate professor at the Victoria University of Wellington, Aotearoa, New Zealand.Credit: Chevron Hassett for Nature

The wharenui behind me in the photograph is in the heart of Victoria University of Wellington, where I lead the school of Māori studies. The detailed carvings, paintings and weavings are a library of traditional knowledge and understanding. The tongues poking from the carved faces on the meeting house might look fierce, but the Māori primarily had an oral culture, and the tongue symbolizes knowledge. The bigger the tongue, the more history, narrative and knowledge there is.I am Māori, and descend from the Ngāti Porou tribe. I research the nexus of Māori knowledge and Western science, and how we can draw the best from both knowledge systems to resolve environmental issues.In 2016, the town of Havelock North suffered a disease outbreak caused by livestock faeces seeping into groundwater. We aim to prevent a recurrence through a better understanding of groundwater and springs. Before the affected area began to be drained for agriculture in the 1870s, it was swampland, and Māori people travelled on the waterways. We might find written reports on spring flow going back 70 years, but Māori knowledge can go back nearly 1,000 years. We are looking at ways to access the knowledge captured in carvings and oral histories — mainly by talking to people who could point out features such as where they swam as a child or gathered eels or cress — to tell us where water once flowed.Another project looks at marine heatwaves, including changes in ocean currents due to climate change. Māori ancestors journeyed across these seas. There is knowledge of ocean currents there, if we can unlock it.In the geometric panels in the photograph, the white triangular ‘teeth’ symbolize strength though unity. I think of Māori knowledge as helping to constrain the scientific data so that they can make better predictions. We want to get to a place where the wider research community realizes that we can’t solve these climate problems with one knowledge system alone.

Nature 598, 228 (2021)
doi: https://doi.org/10.1038/d41586-021-02697-y

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Bioactive composition analysisThe main bioactive components in the three products are listed in Table 1. The main chemical constitutes of DL and LT were quite similar; although significant differences were noted in indicators such as protein (DL  > LT, difference = 8.22, P  DL, difference = 1.79, P  LT, difference = 4.07, P  0.05) of the samples. Among the bioactive constitutes, only POL contents in LT and DL were significantly lower (P  DR (1.90%). Compared with DL and LT, the DR exhibited significantly higher POL, increased by 50.21% (P  More

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