The dorsal abdominal cuticle of Theraphosinae is covered with UrS varying in the shape, size and even in the area number as some taxa may have two separate lateral areas of UrS, e.g., Bistriopelma Kaderka, 2015 and Phrixotrichus Simon, 1889, instead of one dorsal area. Generally, the taxa with UrS of types III, IV and III+IV show higher variability in the shape, size and number of urticating setae patches, in comparison to the taxa with type I setae arranged in one dorsal patch ( Fig 2 ; Bertani & Guadanucci [ 9 ]: Figs 5–12). Theraphosinae with type I setae may have only one to two patterns and differ from the taxa with types III, IV or III+IV, in which another seven patterns were recognised [ 9 ]. The theraphosine genus Magulla Simon 1892 displays another pattern, which consists of type IV setae placed in the central dorsal patch and type III setae present in two separate anterior patches connected by the central patch [ 39 ].

Descriptions of basic urticating setae types and their subtypes

Cooke et al. [6] defined type III setae as 0.3–1.2 mm long setae with thin straight shaft, fine point, barbs along at least one-half the length and considerable variation not only in length but also in the size and density of barbs among setae of this type. As the arrangement of barbs in two opposite rows in Acanthoscurria rhodothele Mello-Leitão, 1923 and in the silhoutte of type III setae presented by Cooke et al. [6] is typical for type I setae and because Pérez-Miles [10] found some differences in the morphology of type III setae in species with the co-occurrence of types I+III and III+IV, we decided to revise and redefine type III setae.

We suggest a new terminology for type I setae with the phylogenetic support of the molecular analyses carried out by Turner et al. [20] and Lüddecke et al. [54], showing taxa with type I setae as monophyletic group, and supported by the findings that were demonstrated in the present study in UrS development during ontogeny in 14 species of Theraphosinae. We consider the UrS that were formerly classified as type III or were considered setae of intermediate morphology between those of types I and III in species possessing both of these types to be ontogenetic derivatives of type I setae as they develop later in the ontogenetic development and they are derived from type I seta morphology. They are further described as subtypes of type I. Other than that, the typology of UrS follows Cooke et al. [6], Marshall & Uetz [5], Pérez-Miles [13] and Perafán et al. [11].

Type I UrS, including their subtypes as morphological repetitions (Fig 3), are generally characterised by the presence of reversed barbs in the midsection and a broad basal end. The basal barbs are present and may be developed (Fig 4A and 4B), reduced (Fig 4C) or strongly reduced (Fig 4D). The connection of the UrS with a supporting stalk, which is a so-called break-off zone, is beneath the basal section of non-reversed barbs; the one exception is in subtype I f with an additional break-off zone between sections B and C1.

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larger image TIFF original image Download: Fig 3. Urticating setae of type I and its subtypes I a , I b , I c , I d , I e and I f . Cross sections (CS) 1–4: arrangement of barbs in cross-sections. The arrows show two rows of opposite reversed denticles on the apical end of section C1. Abbreviations: B = basal section; C1 = central section with well-developed reversed barbs (corresponds with “main barbs” according to Cooke et al. [6]); C2 = central section, which is bare or with two parallel longitudinal rows of short confluent reversed barbs or denticles; A = apical section. https://doi.org/10.1371/journal.pone.0224384.g003

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larger image TIFF original image Download: Fig 4. Urticating setae of Brachypelma smithi, immature male. (A) Basic type I, basal section, connected to the dorsal abdominal surface by a supporting stalk and surrounded by other supporting stalks without urticating setae. Scale bar = 10 μm. (B) Basic type I, basal section with three rows of barbs. Scale bar = 5 μm. (C) Type I, subtypes I c , midsection (below) and basal section (above) with basal barbs reduced in both number and size. Scale bar = 10 μm. (D) Type I, subtype I c , basal section with strongly reduced basal barbs. Scale bar = 10 μm. (E) Basic type I, detail of the midsection with two parallel longitudinal rows of short, confluent reversed barbs in the left lower part of the figure. Scale bar = 5 μm. https://doi.org/10.1371/journal.pone.0224384.g004

Basic type I (Figs 1, 3, 4A, 4B and 4E; Table 2): it is characterised by a shaft with two axial flections and four axial sections (from the basal to the apical end): the basal barbs (section B) are arranged in two opposite, concavely arranged rows; in addition, there is a central third row of longitudinally arranged barbs. The following section (section C2, see Fig 3), which was formerly described as a bare shaft [6], has two parallel rows of short confluent reversed barbs or denticles in adults (Fig 4E). This section may be bare in the first nymphal stages. The following section (section C1, see Fig 3) carries two opposite rows of reversed barbs, which can be arranged helically, in Reversopelma petersi Schmidt, 2001, with 1–4 distal pairs of non-reversed barbs. The tapering apical section (section A, see Fig 3) has only two rows of small reversed denticles. Length of setae: 0.21–0.60.

Subtype I a (Fig 3; Bertani [47]: Fig 5; Table 3): it differs from the basic type I in the absence of the section C2; a third central row of basal barbs or denticles is present. Length of setae: 0.37–0.67. Length of reversed barbs: 0.005–0.030.

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larger image TIFF original image Download: Fig 5. Urticating setae of type I and its subtypes I b and I d . (A) Aenigmarachne sinapophysis, male holotype. Type I, subtype I b , basal section with barbs reduced both in number and size. (B) Aphonopelma seemanni, male. Type I, subtype I b , midsection with three rows of reversed barbs. (C) Reversopelma petersi, male. Type I, distal end of the midsection (section C1) with a few pairs of non-reversed barbs. (D) Citharacanthus longipes, female. Type I, subtype I d , detail of the basal section and section C2. Scale bar = 10 μm. https://doi.org/10.1371/journal.pone.0224384.g005

Subtype I b (Figs 3, 5A and 5B; Table 4): it is characterised by a nearly straight shaft or a shaft with one axial flection and by three axial sections (from the basal to the apical end): the reduced basal section (section B) carries two opposite rows of barbs that are reduced in length and confluent with the shaft, or the barbs are absent. Section C2 is absent. Two rows of opposite reversed barbs on section C1 can be complemented by a third row in the proximal part. The tapering apical section (section A) carries denticles arranged in two opposite rows. Length of setae: 0.27–0.63. Length of reversed barbs: 0.007–0.011.

Subtype I c (Figs 3, 4C and 4D; Pérez-Miles [10]: Fig 6, basal section; Table 5): it is characterised by a long, almost straight shaft or a shaft with one axial flection and three axial sections (from the basal to the apical end): the reduced basal section B carries short barbs, which strongly adhere to the shaft, or the barbs are absent. Section C2 is absent. The following central section, C1, carries thin and long reversed barbs, which are less dense than in the other subtypes, arranged in two longitudinal rows, and complemented by additional one or two rows in the proximal part only. The tapering apical section (section A) carries reversed denticles arranged in two opposite rows. This subtype is clearly longer than the other type I subtypes. Subtype I c differs from I b in total length (I c > I b ), lower density of barbs and larger size (in I c 2–6 times as long as in I b ). It was formerly misinterpreted as a type III seta due to the morphological resemblance, but it differs in the incrassate shaft at the basal end, which carries flattened barbs visible at high magnification (approximately 2000×) [10]. This subtype always occurs with basic type I or another subtype of the type I seta. Length of setae: 0.52–1.64. Length of reversed barbs: 0.022–0.050.

Subtype I d (Figs 3 and 5D; Table 6): it is characterised by a shaft with one axial flection and four axial sections (from the basal to the apical end): the basal barbs (section B) are arranged in two opposite, concavely arranged rows, with a central third row of longitudinal barbs. The following section (section C2), has two parallel rows of short confluent reversed barbs or denticles in adults. It is unusually long, with the C2/C1 ratio higher than 3.0 (up to 9.0). Section C1 is basally flexed, with two opposite rows of reversed barbs. Length of setae: 0.31–0.77. This subtype was found in the genera Citharacanthus Pocock, 1901 and Neischnocolus Petrunkevitch, 1925.

Subtype I e (Figs 3 and 6A; Table 7): it is characterised by its very small size. The setae have one axial flection and four axial sections (from the basal to the apical end): the section of basal barbs (section B) carries three rows of barbs that have a T-shape in cross-section. The following cylindrical C2 section is smooth and stout in comparison to the I f subtype. The following short C1 section has two opposite rows of reversed barbs and the tapering apical section (section A) carries two opposite rows of small reversed denticles. Length of setae: 0.11–0.15. Length of reversed barbs: up to 0.011. This subtype was found only in the females of Metriopelma sp. from Margarita Island, Venezuela.

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larger image TIFF original image Download: Fig 6. Urticating setae of type I and its subtypes I e and I f . (A) Metriopelma sp., female from Margarita Island, Venezuela. Type I, subtype I e , midsection C1 with barbs reduced both in length and number, midsection C2 completely bare, smooth and thicker than in the subtype I f . (B) Pseudhapalopus sp., male from Colombia. Type I, subtype I f , with the midsection C2 narrow and flattened, with a rough surface. Section C2 represents a zone in which the seta breaks off. Scale bar = 20 μm. https://doi.org/10.1371/journal.pone.0224384.g006

Subtype I f (Figs 3 and 6B; Table 8): it is characterised by a smaller size and an extremely narrow and flattened shaft in section C2, which has a rough surface. The setae have two axial flections and four axial sections (from the basal to the apical end): section B is arranged in two opposite rows, and the third central row of longitudinal basal barbs is present. Section C2 represents a zone in which the distal part of the seta breaks off even more easily than the seta breaks off from a supporting stalk. The following section, C1, is curved and has two opposite rows of reversed barbs; the tapering apical section (section A) has two opposite rows of reversed denticles. Length of setae: 0.18–0.21. Length of reversed barbs: up to 0.011. This subtype was found only in a juvenile male of Pseudhapalopus sp. from Colombia.

Type II UrS (Figs 1, 7A–7E, 8 and 9; Table 9): it is characterised by a stout and almost straight shaft with a basal and apical penetrating tip and with two axial sections (from the basal to the apical end): the basal section is equipped with short barbs or scale-like barbs (Fig 9C), which are restricted to the basal third only, with the exception of the UrS of Caribena versicolor (Walckenaer, 1837) and C. laeta (C. L. Koch, 1842) [14], whose barbs are longer and scattered along the whole shaft except the apex. The tapering apical section is bare. The morphology of UrS in two studied nymphs of C. versicolor (length of carapace (car.) 2.5 and 3.5) (Fig 7B, 7D and 7E) is congruent with that of the adults. The basal tips of the tested Aviculariinae were slightly curved upwards.

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larger image TIFF original image Download: Fig 7. Urticating setae of type II. (A) Avicularia sp., female from Beni province, Bolivia. Type II, basal section with a supporting stalk. Scale bar = 20 μm. (B) Caribena versicolor, juv. (car. 3.5). Type II, basal section. Scale bar = 2 μm. (C) Caribena versicolor, male. Type II, apical section. Scale bar = 10 μm. (D) Caribena versicolor, juv. (car. 2.5). Type II, midsection. Basal part on the left. Scale bar = 5 μm. (E) Caribena versicolor, juv. (car. 3.5). Type II, apical section. The basic morphology of urticating setae in juveniles is identical to mature specimens, but the barbs are less developed. Scale bar = 10 μm. Abbreviations: juv. = juvenile specimen; car. = length of carapace. https://doi.org/10.1371/journal.pone.0224384.g007

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larger image TIFF original image Download: Fig 8. Urticating setae of type II in a female of Antillena rickwesti. (A) Type II, basal section with a supporting stalk, the apical section is bare. (B) Seta of intermediate morphology between body setae and type II, basal section with a supporting stalk. The arrow shows a precursor of the basal tip. Scale bar = 10 μm. https://doi.org/10.1371/journal.pone.0224384.g008

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larger image TIFF original image Download: Fig 9. Urticating setae of type II and body seta of an immature female of Iridopelma hirsutum. (A) Type II, detail of the midsection. Scale bar = 5 μm. (B) Body seta, detail of the midsection. Scale bar = 5 μm. (C) Type II, detail of the basal section with a supporting stalk. Scale bar = 10 μm. https://doi.org/10.1371/journal.pone.0224384.g009

Type II urticating setae are typical for some Aviculariinae genera such as Antillena Fukushima & Bertani, 2017, Avicularia Lamarck, 1818, Caribena Fukushima & Bertani, 2017, Iridopelma Pocock, 1901, Pachistopelma Pocock, 1901, Typhochlaena C. L. Koch, 1850, and Ybyrapora Fukushima & Bertani, 2017 [14,15].

In most of Aviculariinae with type II setae, the UrS can be dispersed through direct contact with the intruder [6,7], or may be airborne (recorded in C. versicolor; [8]). The shafts of airborne setae carrying well-developed barbs are much longer and narrower than those of the setae released by direct contact [8]. The length/width ratio is approximately three times higher for airborne setae than for setae released by direct contact [8]. Bertani et al. [8] supposed that the airborne setae represent a homoplastic character shared with Theraphosinae and are a derived character for the Aviculariinae genera Avicularia, Iridopelma and Pachistopelma. According to Cooke et al. [6] and Bertani & Marques [7], the penetrating tip is located basally near the supporting stalk; the mechanism of release of type II UrS was described and determined by the latter authors. In the present study we observed that type II setae also penetrated the target through their apical tips in two Aviculariinae (females of Avicularia sp. from Bolivia and Pachistopelma bromelicola Bertani, 2012). Both females were stimulated and irritated by the oval piece of polystyrene hold in tweezers. The distribution area of type II setae covers the northern part of South America, including some Caribbean islands (Kaderka [57]: Fig 1).

The setae of intermediate morphology between the body setae and the type II urticating setae (Fig 8B), which were found in Iridopelma hirsutum Pocock, 1901 and Antillena rickwesti (Bertani & Huff, 2013), provide another evidence that type II UrS evolved from the body setae (Figs 9B and 10). These setae have a scale morphology restricted to the basal end only and are up to one fifth of the seta length. The apical section is densely covered with short barbs arranged in many longitudinal rows. In addition, they are nearly the same length as the body setae. Length of UrS: 0.45–1.66. Length of setae of intermediate morphology: 0.48–0.51. Length of body setae: 0.47–0.53.

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larger image TIFF original image Download: Fig 10. Body setae of a female of Antillena rickwesti. (A) Body seta. Scale bar = 50 μm. (B) Detail of the apical section. Scale bar = 10 μm. (C) Detail of the basal section with a supporting stalk. Scale bar = 10 μm. https://doi.org/10.1371/journal.pone.0224384.g010

Type III UrS (Figs 1, 11A–11D, 12A and 12B; Table 10): the seta is characterised by an almost straight shaft and by two axial sections: the long basal section has reversed barbs that are usually arranged in 4–5 longitudinal rows, the basal end of the shaft is slightly tapered. The connection of the seta with a supporting stalk is between the tips and basal ends of the basalmost reversed barbs. The short, tapering apical section lacks barbs but has reversed denticles. Length of setae: 0.07–1.25. Length of reversed barbs: 0.003–0.013. Setae of this type are usually arranged in one dorsal patch (most Theraphosinae) or in two dorsolateral patches, e.g., in Phrixotrichus, Bistriopelma, Magulla [39], and Tmesiphantes hypogeus Bertani, Bichuette & Pedroso, 2013 [58].

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larger image TIFF original image Download: Fig 11. Urticating setae of type III. (A) Cyriocosmus perezmilesi, juv., a stage of the first nymph (according to Foelix [56]). Ontogenetic precursor of type III urticating setae, detail of the basal sections with non-reversed barbs. The arrangement of barbs in this stage is congruent with that of body setae presented by Bertani & Guadanucci [9]: Fig 25. Scale bar = 10 μm. (B) Hapalopus sp., juv. (car. 1.8), Lara State, Venezuela. Type III, supporting stalks in the basal sections marked by arrows. Scale bar = 5 μm. (C) Hapalopus sp., juv. (car. 1.8), Lara State, Venezuela. Type III urticating seta with unusually developed basal barbs, detail of the basal section. Scale bar = 5 μm. (D) Hapalotremus sp., a female from Peru. Type III, the apical (lower urticating seta) and the basal section (upper urticating seta). Scale bar = 20 μm. Abbreviations: juv. = juvenile specimen; car. = length of carapace. https://doi.org/10.1371/journal.pone.0224384.g011

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larger image TIFF original image Download: Fig 12. Urticating setae of type III and IV. (A) Theraphosa blondi, female. Long seta of type III, basal section, total length 0.28–0.32. Scale bar = 20 μm. (B) Theraphosa blondi, female. Short seta of type III, total length 0.07–0.08. Scale bar = 20 μm. (C) Chromatopelma cyanopubescens, female. Type IV with reversed barbs in the basal section and reversed denticles in the apical section. Scale bar = 10 μm. (D) Chromatopelma cyanopubescens, female. Type IV, basal end, the arrow shows the connection to a supporting stalk, located between the last two basal barbs. Scale bar = 5 μm. https://doi.org/10.1371/journal.pone.0224384.g012

Type IV UrS (Figs 1, 12C and 12D; Table 11): the seta is characterised by a small size (up to 0.20) and a bent shaft. The basal section has strong reversed barbs (the diameter of the barbs at the basal end is comparable to the diameter of the shaft at the connection site). The central section has small reversed barbs arranged asymmetrically along the shaft. The tapering apical section has two opposite rows of reversed denticles. The connection of the seta with a supporting stalk is between the tips and basal ends of the well-developed basalmost reversed barbs. Length of setae: 0.08–0.21. Length of reversed barbs: 0.004–0.009. According to Bertani & Guadanucci [9], type IV UrS can be better characterised by the barbs pointing towards the convex side of the seta. The occurrence of this type is restricted to the South American Theraphosinae only (see Table 1).

Type V UrS (Figs 1, 13A and 13B; Table 12): the seta is characterised by an almost straight shaft and by two axial sections. The long basal section (80–90% of the seta length) has barbs arranged asymmetrically along the shaft; the angle between the shaft and the barbs is approximately 10–30° and the barbs are more confluent on the base. The short, tapering apical section is bare. Type V setae are inserted in the sockets on the palpal cuticle, and supporting stalks are absent. The seta break-off zone is inside the socket. Length of setae: 0.55–0.67. Length of barbs: 0.009–0.011. This type of urticating setae is located distally on the prolateral face of the palpal femora of Ephebopus spp. [59,60]. The setae are densely packed and uniformly arranged (Fig 13). This unique morphological characteristic is considered a generic characteristic of Ephebopus [59]. According to Bertani & Marques [7], there is no reason to consider this type of setae as homologous to the abdominal setae in Aviculariinae or Theraphosinae.

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larger image TIFF original image Download: Fig 13. Palpal urticating setae of type V in an immature female of Ephebopus cyanognathus. (A) Type V urticating setae connected with the palpal surface by insertion sockets. Scale bar = 10 μm. (B) Prolateral face of the right palp with a limited area of insertion sockets without urticating setae. Scale bar = 100 μm. https://doi.org/10.1371/journal.pone.0224384.g013

Type VI UrS (Figs 1, 14A–14E and 15; Pérez-Miles [13]: Figs 1–4; Table 13): this seta, with an almost straight shaft, is connected to the abdominal surface by cylindrical supporting stalks (Fig 14A). The barbs are subbasally short and more confluent to the shaft (for approximately 10% of the seta length) and longer and more protruding on the rest of the seta (the angle between the shaft and the barbs is approximately 30°). The apical section (approximately 10% of the seta length) can be bare (Pérez-Miles [13]: Figs 1–4) or can have well-developed protruding barbs reaching the apex, and causing the absence of the apical tip. Reversed barbs are absent on type VI setae. Length of setae: 0.64–1.21. Length of barbs (measured in apical region): 0.007–0.010. The setae can be arranged in one dorsomedial patch, two dorsal paramedian patches, or in two lateral patches [35]. This type was found only in the Mexican genus Hemirrhagus, with the exception of some troglobitic species [35]. Type VI setae are distinguished from the very similar type II setae by the presence of long barbs in the apical half. The morphology of the basal half is congruent.

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larger image TIFF original image Download: Fig 14. Urticating setae of type VI. (A) Hemirrhagus papalotl, female. Type VI, two midsections with short, more confluent barbs, apical section with longer and more protruding barbs, basal section with a supporting stalk. Scale bar = 10 μm. (B) Hemirrhagus eros, female. Type VI, the basal section with a socket for supporting stalk. Scale bar = 5 μm. (C) Hemirrhagus papalotl, immature male. Type VI, detail of the basal section. Scale bar = 5 μm. (D) Hemirrhagus papalotl, immature male. Type VI, detail of the midsection. Scale bar = 5 μm. (E) Hemirrhagus papalotl, immature male. Type VI, detail of the apical section. Scale bar = 2 μm. https://doi.org/10.1371/journal.pone.0224384.g014

Type VII UrS (Perafán et al. [11]: Figs 2 and 8): the seta is straight and has small reversed subtriangular barbs or denticles along the entire shaft. These barbs are not homogenous in size or density and they are longer in the basal part. A small oval patch of lanceolated barbs is located in the apical quarter near the penetrating tip. The lanceolated barbs are longer, broader and less acute than the reversed barbs. The length/width ratio of the shaft is approximately 34:1. The setae are connected to the abdominal surface by thinner stalks. Type VII seta resembles type II but differs in the presence of the patch of lanceolated barbs and in the presence of reversed barbs scattered along the shaft [11]. These setae were found exclusively in Kankuamo [11].

The urticating setae types found at the examined specimens are listed in Table 14.