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Comparative morphology of stridulating setae of Theraphosinae (Araneae: Theraphosidae)
Zoologischer Anzeiger 283 (2019) 58e68
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Research paper
Comparative morphology of stridulating setae of Theraphosinae (Araneae: Theraphosidae) Paulo Leite Guadanucci Arthur Galleti-Lima*, Jose ~o Paulo State University (UNESP e Rio Claro), Rio Claro, Sa ~o Paulo, Brazil Department of Zoology, Institute of Biosciences, Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2019 Received in revised form 25 June 2019 Accepted 30 August 2019 Available online 7 September 2019
Theraphosid spiders are repleted with cuticular structures that evolved into distinct morphologies and developed different functions. The stridulatory apparatus present in the subfamily Theraphosinae is composed by a group of setae and opposed organs (ridged surface) that emit sound through friction. Only sixteen genera of Theraphosinae spiders were reported with stridulatory setae: Acanthoscurria, Aguapanela, Brachypelma, Citharacanthus, Cyrtopholis, Grammostola, Hemirrhagus, Lasiodora, Longilyra, Nhandu, Theraphosa, Pamphobeteus, Phormictopus, Proshapalopus, Pterinopelma and Vitalius. Besides these stridulating setae, other cuticular structures have been traditionally used in Theraphosinae taxonomy: the plumose scopula on the lateral faces of femora I and IV. However, the ultrastructure of these setae and scopulae was never studied in detail. Here, we analyzed the diversity of cuticular structures on coxae, trochanters and femora of Theraphosinae spiders using SEM. Beside the description of plumose and claviform stridulating setae on femora, we established five distinct variations of stridulatory setae found on coxae and trochanters and report their presence for the genus Megaphobema, Crassicrus and Cotztetlana for the first time. © 2019 Elsevier GmbH. All rights reserved.
Corresponding Editor: P. Michalik Keywords: Tarantula Ultrastructure Stridulation SEM
1. Introduction The spider family Theraphosidae is currently divided in eleven subfamilies with the neotropical subfamily Theraphosinae as the most diverse group. The presence of setae, or groups of setae, forming structures named lyra, stridulatory apparatus and scopulae, mainly on basal articles of legs and palps, such as coxae and trochanters is very common within Theraphosidae (Raven 1985; Smith 1990). Many group diagnoses are based on the possession of these distinct setae, and some of these spiders are notorious for producing sound using some body setae (Uetz & Stratton 1982; Marshal et al. 1995). This phenomenon known as stridulation (Pocock 1895) is the result of the friction of these structures, which are often located on coxae and trochanters of legs and palps (Marshall et al. 1995). Some authors have suggested sexual or defensive roles for such stridulation. (Legendre 1963; Uetz & Stratton 1982; Marshall et al. 1995). According to Uetz & Stratton (1982), the stridulation occurs by the scratching of structures located on opposite faces, producing
* Corresponding author. E-mail addresses: [email protected] [email protected] (J.P.L. Guadanucci). https://doi.org/10.1016/j.jcz.2019.08.010 0044-5231/© 2019 Elsevier GmbH. All rights reserved.
(A.
Galleti-Lima),
vibrations and, in some cases, audible sounds for humans. The knowledge about occurrence and morphology of stridulating setae in Theraphosinae is scarce and limited to taxonomic studies without an inter-generic comparative approach. The majority of the taxonomic literature refers to the occurrence of these structures, however, SEM data are present in just a few of them. To date, the following Theraphosinae genera were reported with stridulating setae: Acanthoscurria Ausserer, 1871; Aguapanela n & Cifuentes, 2015; Brachypelma Simon, 1891; Cyrtopholis Perafa Simon, 1892; Citharacanthus Pocock, 1901; Grammostola Simon, 1892; Hemirrhagus Simon, 1903; Lasiodora C. L. Koch, 1850; Longilyra Gabriel, 2014; Nhandu Lucas, 1983; Pamphobeteus Pocock, ~o, 1901; Phormictopus Pocock, 1901; Proshapalopus Mello-Leita 1923; Pterinopelma Pocock, 1901; Theraphosa Therell, 1870 and Vitalius Lucas, Silva & Bertani, 1993. However, not all of these representatives had their coxae and/or trochanter examined under the SEM, which is essential for a proper recognition of such setae. Besides stridulating setae on coxae and trochanter, Theraphosinae spiders are replete with other setae types of taxonomic relevance. According to Raven (1985), the presence of scopulae on the retrolateral face of femur IV is synapomorphic for the subfamily Theraphosinae. The occurrence of these scopula e was also used as a rez-Miles et al. (1996) and Peraf character by Pe an et al. (2016) in
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
their phylogenetic hypotheses of Theraphosinae intrarelationships. However, contrary to Raven (1985), it was shown that many Theraphosinae representatives lack the femoral scopula. According to Peraf an et al. (2016), the following genera are characterized with the presence of femoral scopula: Acanthoscurria, Cyrtopholis, Eupalaestrus Pocock, 1901, Lasiodora, Megaphobema Pocock, 1901, Metriopelma Becker, 1878, Nhandu, Pamphobeteus, Phormictopus, Schizopelma F. O. Pickard-Cambridge, 1897, Sericopelma Ausserer, 1875, Theraphosa, Vitalius e Xenesthis Simon, 1891. In this study, we performed a comparative ultrastructural analysis of stridulatory setae of Theraphosinae representatives. Furthermore, we present an inventory of these setae and propose a standard terminology for Theraphosinae taxonomy complementing the descriptions in Galleti-Lima & Guadanucci (2018). 2. Material and methods The material examined belongs to the following scientific col~o Paulo, Brasil (IBSP) e A. Brescovit; lections: Instituto Butantan, Sa ^ncias Naturais, Fundaça ~o Zoobot^ Museu de Cie anica do Rio Grande n Nacional de do Sul, Porto Alegre, Brasil (MCN) e R. Ott; Coleccio cnidos UNAM, Me xico DF, Me xico (CNAN) e O. F Ballve ; Centro Ara ^ncias Exatas e da Natureza, Universidade Federal da Paraíba, de Cie ~o Pessoa, Brazil (CCEN) e M. B. da Silva; Coleça ~o Aracnolo gica Joa Diamantina, Rio Claro, Brazil (CAD) e J. P. L Guadanucci; Personal n Garcia. Collection Alayo Material examined (geographic coordinates between parentheses indicate the location of the municipality, obtained from Google earth): ~o, 1923: 1 _, 1 \ (CAD Acanthoscurria gomesiana Mello-Leita 472). Ami sp.: 1 _, (MPEG 02-220), 1 \, (MPEG 02-088). Aphonopelma anitahoffmannae Locht et al., 2005: 1 _, 1 \, (CNAN 3047). Brachypelma smithi F. O. Pickard-Cambridge, 1897: 1 _, Exoesqueleton. Bumba sp.: 1 _, (IBSP 151865). Catanduba flavohirta Simon, 1889: 1 _, (IBSP 126901). Catanduba sp.: 1 _, (CAD). Cotztetlana sp.: 1 \, Exoesqueleton. Crassicrus sp.: 1 \, Exoesqueleton. rez-Miles, 1998: 1 _, (CAD), 1 \, (CAD). Cyriocosmus chicoi Pe Cyrtopholis sp.: 1 _, (IBSP), 1 \, (IBSP). Davus sp.: 1 _, (CNAN 3515). Euathlus sp.: 1 _, (IBSP 3817-A). Eupalaestrus sp.: 1 _, (IBSP 4200). Grammostola sp.: 1 _, (IBSP 2427), 1 \, (IBSP 2427). Hapalopus rez-Miles, 1998.: 1 _, (MPEG 019077), 1 \, (MPEG butantan Pe rez-Miles & Locht, 2003: 1 _, 019081). Hemirrhagus papalotl Pe (CNAN). Hemirrhagus sp.: 1 \, (CNAN-Ar. 010269). Hemirrhagus billsteelei Mendoza & Francke, 2018: 1 _, (CNANAr. 010274). Hemirrhagus kalebi Mendoza & Francke, 2018: 1 _, (CNAN). Homoeomma montanum Mello-Leit~ ao, 1923: 1 _, (IBSP 4685). Kochiana sp.: 1 _, (IBSP 11716). Lasiodora klugi C. L. Koch, 1841.: 1 _, (CCEN. 181). Magulla obesa Simon, 1892: 1 _, (IBSP 8367), 1 \, rez-Miles, Miglio & Bonaldo, 2006: (CAD). Megaphobema teceae Pe 1 _, (MPEG 005233). Nhandu cerradensis Bertani, 2001: 1 _, (IBSP 11847); 1 \, (IBSP 13971). Pamphobeteus augusti Simon, 1889: 1 _, n Garcia. Plesiopelma sp.: 1 \, ExoesPersonal Collection Alayo queleton. Phormictopus cautus Ausserer, 1875: 1 _, (IES, CZAAC ~o, 1929: 1 _, 3.4244). Proshapalopus multicuspidatus Mello-Leita (CAD 094). Pterinopelma felipeleitei Bertani & Leal, 2016: 1 _, (CAD 441), 1 \, (CAD 584). Pterinopelma sazimai Bertani, Nagahama & Fukushima, 2011: 1 _, (CAD), 1 \ (CAD). Pterinopelma vitiosum Keyserling, 1891: 1 _, (MCN 22145), 1 \, (MCN 22102). Theraphosa blondi Latreille, 1804: 1 _ (MPEG ARA 000176), 1 \, (MPEG 007558). Tmesiphantes sp.: 1 _, (CAD). Vitalius sp.: 1 _, (CAD). 2.1. Scanning Electron Microscopy (SEM) Coxae, trochanters and femora were dissected and manually cleaned with brush. Then the material was ultrasonic cleaned,
59
using a water and detergent solution. After ultrasonic cleaning, the material underwent dehydration in graded ethanol (70%, 80%, 90% and absolute ethanol e 60 min in each concentration) and were critical-point dried. The material was glued to stubs with doublesided copper tapes, sputter-coated with gold and examined in a Hitachi TM- 1000 Scanning Electronic Microcopy, at the Electronic Microscopy Laboratory of the Institute of Biosciences of Sao Paulo ~o Paulo, Brazil. State University, campus Rio Claro, Sa 2.2. Seta types and morphometry All setae present on the opposite faces of coxae, trochanters and femora with distinct morphological features different from the surrounding regular setae were investigated. We considered regular setae as those present also on other body parts, such as carapace, sternum, abdomen, spinnerets and other leg articles. The lengths of setae were measured with the software ImageJ Fiji 32-bit (Schindelin et al. 2012). Whenever possible, five setae were measured per specimen. Pictures were edited for brightness/ contrast corrections and grouped in plates with Photoshop CS6. 3. Results In order to apply a standardized description for stridulatory rez-Miles et al. (2005) and Galleti-Lima & setae, we followed Pe Guadanucci (2018), who established morphological patterns and terminology for this type of setae. Unfortunately, we did not have specimens of the genera Longilyra and Citharacanthus, and therefore used original descriptions for our analysis. Table 1 shows the distribution, morphology and morphometric data of stridulatory setae examined in representatives of Theraphosinae. Those specimens included in the list of material examined but not present in any table, figure or through the text henceforth, do not possess any type of stridulating setae. 3.1. Pilose stridulating setae These setae are only present in Hemirrhagus sp. and are characterized by having a smooth base with modestly long barbs extending from the shaft region to the apex. They differ from the plumose stridulating setae (Peraf an et al. 2015), by the lack of grooves at the apex surface (Figs. 9e12). The pilose stridulating setae arrangement can be seen in Fig. 40. In addition, we found two variations, thin (Figs. 9e10), restricted to coxae, and thick (Figs. 11e12), restricted to trochanters. 3.2. Sectioned stridulating setae The sectioned stridulating setae are characterized by the robust appearance and the smooth base that extends up to approximately the mid length, where small barbs are arranged in parallel lines and extend to the apex, giving it the appearance of longitudinal sectioned regions marked by conspicuous longitudinal furrows (Figs. 1e2). This seta type was observed in Aphonopelma annitahoffmannae, H. billsteelei and H. kalebi. The arrangement of these setae is shown in Figs. 31, 34, 41 and 42. 3.3. Spatuliform stridulating setae These setae are characterized by having a smooth base and a mid region with a membrane-like projection covered with small short and thin beards, extending to the apex (Figs. 13e14). They were found in H. billsteelei and H. kalebi and their arrangement can be observed in Figs. 41 and 42.
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A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
Table 1 Distribution, morphology and morphometry of stridulatory setae of Theraphosinae genera. Measurements are in micrometers (mm); SD ¼ standard deviation; n ¼ sample size, () ¼ not examined. The spiniform setae of the genus Pamphobeteus is common in other Theraphosidae spiders. Stridulating setae type
Genera
Figures setae
Sectioned
Aphonopelma
1e2, 31 and 32
Sectioned Sectioned
Citharacanthus Hemirrhagus billsteelei
Schmidt (2003): fig. 190, 191, 293 and 294; Fig. 34 Figs. 1e2 and 53
Sectioned
Hemirrhagus kalebi
Figs. 1e2 and 54
Spiniform
Cotztetlana
Figs. 3 and 35
Spiniform
Theraphosa
Galleti-Lima & Guadanucci (2018): fig. 14; Fig. 57
Plumose
Crassicrus
Figs. 4 and 36; Seta detail in Galleti-Lima & Guadanucci (2018): figs. 19e22
Plumose
Megaphobema
Figs. 5 and 45
Plumose
Acanthoscurria
Galleti-Lima & Guadanucci (2018): 11, 12 and 13; Fig. 29
Plumose Plumose
Aguapanela Lasiodora
Peraf an et al. (2015): figs 1e12; Fig. 30 Galleti-Lima & Guadanucci (2018): fig. 8; Fig. 43
Plumose
Nhandu
Galleti-Lima & Guadanucci (2018): fig. 9; Figs. 46 and 47
Plumose
Proshapalopus
Galleti-Lima & Guadanucci (2018): fig. 6; Fig. 50
Plumose
Pterinopelma
Galleti-Lima & Guadanucci (2018): fig. 7; Figs. 51e56
Plumose
Vitalius
Galleti-Lima & Guadanucci (2018): fig. 10; Fig. 58
Claviform Claviform
Phormictopus Acanthoscurria
Figs. 6 and 49; Seta detail in Galleti-Lima & Guadanucci (2018): figs. 15e18 Galleti-Lima & Guadanucci (2018): figs. 1; and 29
Claviform
Brachypelma
Galleti-Lima & Guadanucci (2018): fig. 3; Fig. 33
Claviform
Cyrtopholis
Galleti-Lima & Guadanucci (2018): fig. 2; Fig. 37
Claviform
Theraphosa
Galleti-Lima & Guadanucci (2018): fig. 4; Fig. 57
Rough
Grammostola
Figs. 7e8 and 39
Pilose thin
Hemirrhagus sp.
Figs. 9e10 and 40
Pilose thick
Hemirrhagus sp.
Figs. 11e12 and 40
Spatuliform
Hemirrhagus billsteelei
Figs. 13e14 and 41
Spatuliform Spiniform (common variation) Velvet
Hemirrhagus kalebi Pamphobeteus
Figs. 13e14 and 42 Figs. 15e16 and 48
Lasiodora
Galleti-Lima & Guadanucci (2018): figs. 5 and 8; Fig. 43
Liriform
Longilyra
Gabriel (2014): figs 1e3; Fig. 44
Average Length e mm (±SD) _
\
271,4 (n ¼ 1) e 503.0 (±503.0) (n ¼ 3) 327.6 (±19.2) (n ¼ 3) e
e
938.7 (±178.2) (n ¼ 3) e 517.4 (±99.0) (n ¼ 4) 584.8 (±79.4) (n ¼ 4) e 817.3 (±130.7) (n ¼ 5) 413.2 (±60.1) (n ¼ 3) 375.2 (±81.3) (n ¼ 5) 610.8 (±147.8) (n ¼ 3) 616.1 (±67.5) (n ¼ 4) 1147.1 (n ¼ 1) 594.7 (±203.1) (n ¼ 5) 577.5 (±268.2) (n ¼ 5) 425.9 (±43.1) (n ¼ 2) 420.9 (n ¼ 1) 682.3 (±140.3) (n ¼ 3) 368.7 (±182.5) (n ¼ 4) 446.1 (±181.0) (n ¼ 2) 627.9 (±151.5) (n ¼ 5) 181.0 (n ¼ 1) e 1056.2 (±150.9) (n ¼ 3) e
Fig. 1e2. Sectioned stridulating setae of Aphonopelma annitahoffmannae. 1. Overview. 2. Apex. Images: A. Galleti-Lima.
e e e 433.3 (±95.3) (n ¼ 4) 668.3 (±296.2) (n ¼ 5) 529.7 (±60.1) (n ¼ 3) e 538.1 (±130.2) (n ¼ 4) e e 566.5 (±70.0) (n ¼ 5) e 480.178 (±63.7) (n ¼ 2) e e 259.2 (±98.2) (n ¼ 5) e 316.7 (±37.9) (n ¼ 4) 1039.4 (±182; 6) (n ¼ 2) 685.8 (±244.6) (n ¼ 4) e e e e e e e
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61
Fig. 3e6. Types of stridulating setae. 3. Spiniform stridulating setae of Cotztetlana. 4. Plumose stridulating setae of Crassicrus (arrows showing the stridulating setae). 5. Plumose stridulating setae of Megaphobema. 6. Claviform stridulating setae of Phormictopus. Images: A. Galleti-Lima.
Fig. 7e8. Rough stridulating setae of Grammostola sp. 7. Overview. 8. Apex. Images: A. Galleti-Lima.
Fig. 9e10. Pilose stridulating setae (thin variation) of Hemirrhagus sp. 9. Overview. 10. Apex. Images: A. Galleti-Lima.
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Fig. 11e12. Pilose stridulating setae (thick variation) of Hemirrhagus sp. 11. Overview. 12. Apex. Images: A. Galleti-Lima.
Fig. 13e14. Spatuliform stridulating setae of Hemirrhagus billsteelei. 13. Overview. 14. Apex. Images: A. Galleti-Lima.
Fig. 15e16. Tactile spiniform setae of Pamphobeteus augusti. 15. Overview (arrows showing the tactile setae). 16. Apex. Images: A. Galleti-Lima.
3.4. Rough stridulating setae The rough stridulating setae type is reported for Grammostola, being characterized by a smooth base and a middle region completely covered by short barbs that extend to the apex (Figs. 7e8). The arrangement of these setae in coxae and trochanters are shown in Fig. 39. 3.5. Liriform stridulating setae The description of this type of seta is based on data from Gabriel (2014), as we did not have material available to examine. Their images show parallel disposed setae, which have a feather-like aspect and swollen apex, present in spiders of the genus Longilyra Gabriel, 2014 on the prolateral trochanter of leg I (Fig. 44). The number of setae ranges from 11 to 16 (Gabriel 2014). The terminology for these setae refers to the original description of the genus
(Gabriel 2014), as the term lyra was used by the author to describe the stridulating setae, which are disposed in parallel. Distinct types of stridulating setae may occur on the same individual, as reported by Galleti-Lima & Guadanucci (2018) for the genera Acanthoscurria, Lasiodora and Theraphosa. In the present work, we observed the same organization for H. billsteelei, with spatuliform and sectioned types. Regarding the scopula on femora IV, our ultrastructural analysis revealed plumose stridulating setae on the retrolateral side in representatives of the genera Acanthoscurria (Fig. 29), Eupalaestrus (Fig. 38), Lasiodora (Fig. 43), Megaphobema (Fig. 45), Nhandu (Figs. 46e47), Pamphobeteus (Fig. 48), Phormictopus (Fig. 49), Proshapalopus (Fig. 50), Pterinopelma (Figs. 51e56) and Vitalius (Fig. 58). The genera Acanthoscurria, Eupalaestrus, Lasiodora, Nhandu and Vitalius also have femoral scopula (plumose stridulating setae) on prolateral femur I, distinguishing them from the remaining taxa that have femoral scopula on femur IV only. All
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Fig. 17e24. Stridulating setae of femoral scopula. 17. Plumose stridulating setae of the femora I and IV of Acanthoscurria (arrows showing the stridulating setae). 18. Claviform stridulating setae of the palpal femur, legs I, II, III and IV of Brachypelma. 19. Plumose stridulating setae of femora I and IV of Eupalaestrus. 20. Plumose stridulating setae of femora I, II, III and IV of Lasiodora. 21. Plumose stridulating setae of femora I and IV of Megaphobema (arrows showing the stridulating setae). 22. Plumose stridulating setae of femora I and IV of Nhandu. 23. Plumose stridulating setae of the femur IV of Pamphobeteus. 24. Plumose stridulating setae of the femur IV of Phormictopus. Images: A. Galleti-Lima.
investigated specimen are characterized by a less dense femoral scopula on leg I compare to that on femur IV. The genera Brachypelma and Theraphosa also present femoral scopula on the palps (retrolateral) and leg femora I-IV (see different distributions in Figs. 33 and 57), but these are composed by claviform stridulating setae. Genera with stridulating setae on its femora are listed in Table 2, with seta type, occurrence and morphometric data. Figs. 29e58 show the distribution of femoral scopula, as well as the stridulating apparatus on coxae and trochanters.
4. Discussion All stridulatory setae examined herein, and also those described in Galleti-Lima & Guadanucci (2018) among numerous other studies (e.g. Bücherl 1951; Gerschman & Schiapelli 1966; Marshall rez-Miles et al. 2005; Rudloff & et al. 1995; Bertani 2001; Pe Weinmann 2010), share the following features: (1)- restricted to opposite faces (coxae, trochanters and femura), instead of spreading through the body, or opposed to another structure (e.g.
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Fig. 25e28. Stridulating setae of the femoral scopula. 25. Plumose stridulating setae of the femur IV of Pterinopelma (arrows showing the stridulating setae). 26. Plumose stridulating setae of femur IV Proshapalopus. 27. Claviform stridulating setae legs I, II, III and IV femora of Theraphosa. 28. Plumose stridulating setae of femora I and IV of Vitalius (arrows showing the stridulating setae). Images: A. Galleti-Lima.
other setae, ridges) to warrant friction for producing vibration; (2) rigidness, resisting attempts to bent with a needle or tweezers; (3) surface with rough texture, with barbs or ridges. All these features seem to be a requirement to produce vibration, which in some cases are audible hissing sound for humans. Many other regular body setae also have barbs, such as the tactile setae, which in Theraphosidae are characterized by the insertion on a socket and very short barbs, or the scale-like setae, which are very distinct from other setae as their base strongly bent (Ramirez 2014). These types of setae, can participate of the stridulation, but apparently not rez-Miles et al. 2005; are necessarily obligatory in the process (Pe Ferretti et al. 2011). Moreover, groups of stridulatory setae are very distinct from any other setae around them. Although in some cases they are hard to identify under the stereomicroscope, they are easy to distinguish under the SEM. 4.1. Plumose setae Galleti-Lima & Guadanucci (2018) highlighted the importance of the examination of the seta ultrastructure in order to characterize and identify the morphological patterns. Ortiz & Bertani (2005) used the term “plumose seta” to describe the stridulatory apparatus of Phormictopus, examined under the stereomicroscope. Galleti-Lima & Guadanucci (2018) did not have specimens of Phormictopus available for examination and assumed that this seta should be the claviform setae, based on the close evolutionary affinities with Acanthoscurria and Cyrtopholis. The data presented here confirms the claviform condition of Phormictopus stridulating setae. According to Galleti-Lima & Guadanucci (2018), the claviform stridulating setae found in the genus Phormictopus can be considered as an additional generic diagnostic feature, including Brachyn et al. pelma, Acanthoscurria, Cyrtopholis and Theraphosa. Perafa (2015), reported tufts of stridulating setae for spiders of the genus Aguapanela. This new variation of stridulating setae, was named stridulating plumose setae and were found on prolateral face of coxae, trochanters and femora of legs I and II. Galleti-Lima &
Guadanucci (2018) found the same type of setae when investigating these tufts in representatives of Acanthoscurria, Proshapalopus, Pterinopelma, Lasiodora, Nhandu and Vitalius. According to Marroquin (2014) and Mendoza & Francke (2018), some species of the genus Hemirrhagus possess a variety of stridulating setae on lateral faces of trochanters and coxae of legs and palp: Hemirrhagus akheronteus Mendoza & Francke, 2018 (feathery), H. billsteelei (claviform and spiniform), Hemirrhagus rez-Miles & Locht, 2003 (plumose), Hemirrhagus diablo chilango Pe Mendoza & Francke, 2018 (plumose), Hemirrhagus franckei Mendoza, 2014 (spiniform and plumose), H. kalebi (parallel and plumose claviform), Hemirrhagus nahuanus Gertsch, 1982 (spiniform and plumose), Hemirrhagus perezmilesi García-Villafuerte & Locht, 2010 (spiniform and claviform), Hemirrhagus pernix Ausserer, 1875 (spiniform and plumose), Hemirrhagus reddelli Gertsch, 1973 (spiniform, plumose and stridulating setae on femur) and Hemirrhagus sprousei Mendoza & Francke, 2018 (plumose and spiniform). These Hemirrhagus species were not examined under the SEM and the setal names given above are the ones originally proposed by Marroquin (2014) and Mendoza & Francke (2018) not matching with the terminology adopted here and in Galleti-Lima & Guadanucci (2018). In our study, we could only investigate the ultrastructure for three species of Hemirrhagus, which possess the following types of stridulating setae using terminology in GalletiLima & Guadanucci (2018): Hemirrhagus sp. with pilose setae (thin and thick variants); H. billsteelei and H. kalebi with spatuliform setae. The number of stridulating setae found in only three species of Hemirrhagus indicates that the genus possesses a potential for the occurrence of other variations (including new variations), which justifies precise analysis of legs articles under SEM. 4.2. Sectioned stridulating setae Schmidt (2003) reported sectioned stridulating setae for the genus Citharacanthus, on trochanters of palps (retrolateral) and legs I (prolateral) (Fig. 34). Considering the close phylogenetic
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Fig. 29e44. Occurrence of stridulating setae on coxae, trochanters and femora. 29. Acanthoscurria gomesiana. 30. Aguapanela sp. 31-32. Aphonopelma annitahoffmanae. 33. Brachypelma smithi. 34. Citharacanthus sp. 35. Cotztetlana sp. 36. Crassicrus sp. 37. Cyrtopholis sp. 38. Eupalaestrus sp. 39. Grammostola sp. 40. Hemirrhagus sp. 41. Hemirrhagus billsteelei. 42. Hemirrhagus kalebi. 43. Lasiodora klugi. 44. Longilyra sp. Drawing credit: Denis Rafael Pedroso.
n et al., 2016), we expected relationship with Aphonopelma (Perafa that this type of stridulating setae is similar in both genera. However, representatives of the genus Citharacanthus have not been examined under SEM and this hypothesis still needs to be tested.
4.3. Spiniform setae Many authors have used the term “spiniform” to refer to putative stridulating setae, i. e. distinct setae positioned on opposite
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Fig. 45e58. Occurrence of stridulating setae on coxae, trochanters and femora. 45. Megaphobema teceae. 46-47. Nhandu cerradensis. 48. Pamphobeteus augusti. 49. Phormictopus cautus. 50. Proshapalopus multicuspidatus. 51-52. Pterinopelma felipeleitei. 53-54. Pterinopelma sazimai. 55-56. Pterinopelma vitiosum. 57. Theraphosa blondi. 58. Vitalius sp. Drawing credit: Denis Rafael Pedroso.
faces on legs and/or palp articles. In the original description of Pamphobeteus crassifemur and P. grandis, Bertani et al. (2008 e see Figs. 9e12) reported the presence of “spiniform setae” on lateral faces of coxae and trochanters. Bertani et al. (2008) suggested that stridulation in P. crassifemur takes place while the spider sheds urticating setae, when lifting its hind legs. However, a different possibility of a stridulating site in Pamphobeteus could be at femur
IV (plumose retrolateral scopula) (Fig. 48), which might scrap against the anterior lateral faces of abdomen. Although this process of stridulation has never been reported before, our morphological analysis revealed that the femoral scopula is composed of the same type of stridulating setae (plumose or claviform) on coxae and trochanters resembling a potential stridulating apparatus. We found a distinct group of long setae in P. augusti, similar to tactile
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
67
Table 2 Femoral scopulae on Theraphosinae genera: type, occurrence and morphometrics. Stridulating setae
Genera
Femur
Average Length (mm)
Figures
Claviform
Brachypelma
All legs þ palp
Figs. 18 and 33
Claviform
Theraphosa
All legs þ palp
Plumose
Acanthoscurria
I and IV
Plumose Plumose
Eupalaestrus Lasiodora
I and IV All
Plumose Plumose
Megaphobema Nhandu
IV I and IV
Plumose
Pamphobeteus
IV
Plumose
Phormictopus
IV
Plumose
Pterinopelma
Plumose
Proshapalopus
I and IV (P. sazimai) IV (remaining spp) IV
Plumose
Vitalius
I and IV
440.0 (n ¼ 1) 735.3 (±122.8) (n ¼ 4) 695.4 (±131.1) (n ¼ 2) 390.0 (n ¼ 1) 908.2 (±486.3) (n ¼ 4) 1113.7 (n ¼ 1) 731.7 (±117.5) (n ¼ 2) 624.0 (±112.4) (n ¼ 5) 345.8 (n ¼ 1) 559.6 (n ¼ 1) 335.3 (n ¼ 1) 657.1 (±313.2) (n ¼ 5)
setae present on legs as they share the same ultrastructure and the insertion in a socket (Figs. 15e16). We are still far from knowing the whole morphological variants and the process of stridulating in Pamphobeteus. Further detailed micrographies, with the inclusion of a greater diversity of Pamphobeteus species, are still needed to reach a standard terminology. Behavioral studies focused on stridulation may clarify the process of stridulation in Pamphobeteus species. Ferreti et al. (2011: Figs. 4e7, 10, 12) also used the term “spiniform” referring to setae, which were found interspersed with plumose setae, located on opposite faces of coxae and maxillae in Grammostola. They recognized two variants of the same type (short and long) and presented ultrastructural data showing their similarities with regular leg spines: unbarbed, with ridged surface. rez-Miles et al. (2005), the According to Ferretti et al. (2011) and Pe spiniform setae can also participate in the stridulating process, since they are rigid and with a rough surface. Galleti-Lima & Guadanucci (2018: Figs. 14, 27e30) also reported the presence of “spiniform” setae in Theraphosa and Cotztetlana. Gerschamn & Schiapelli (1966) had also reported these in Theraphosa, and they used the term “black spines”. In fact, the spiniform setae in Theraphosa, Grammostola and Cotztetlana are very similar to leg spines and, until behavior studies are carried out, the role in stridulation is questionable. Concerning setal terminology, we promote the use of the term “spiniform” for these specific setae in Theraphosa, Grammostola and Cotztetlana. Those spiders belonging to genera that possess putative stridulating setae, that meet the three conditions for stridulating presented herein, and are notorious for their ability to produce audible sound (e. g. Lasiodora, Theraphosa) making them prime candidates for further investigations. 4.4. Setae on femoral scopula Regarding the femoral scopula across Theraphosinae, the distinct organizations depicted in Figs. 29e58 can be used as diagnostic characters for genera recognition. For example, the close relationship between P. sazimai with Vitalius and Nhandu as recovered in the phylogenetic hypothesis in Galleti-Lima &
Figs. 27 and 57 Figs. 17 and 29 Figs. 19 and 38 Figs. 20 and 43 Figs. 21 and 45 Figs. 22, 46 and 47 Figs. 23 and 48 Figs. 24 and 49 Figs. 25, 51e56 Figs. 26 and 50 Figs. 28 and 58
Guadanucci (2018) and also suggested by Bertani & Leal (2016), receives further support with the presence of femoral scopula on leg I (prolateral). The remaining Pterinopelma species (P. felipeleitei and P. vitiosum) lack femoral scopula on leg I. It might be possible to distinguish the plumose and claviform setae of femoral scopula from the other setae using a stereomicroscope (magnifications above 50). However, the identification of such setae can only reliable be done using SEM. This also explains that many described species lack information concerning the femoral scopula, and other stridulating setae on coxae and trochanter (e.g. Vitalius, Pterinopelma, Nhandu and Proshapalopus). Figs. 59 and 60 show a fe~o, 1923 and a female male Eupalaestrus spinosissimus Mello-Leita P. sazimai with conspicuous femoral scopula, which is depicted in detail (stereomicroscope) in Fig. 61. This femoral scopula can be easily observed, with the help of a stereomicroscope or not, because the setae that constitute this scopula have the same length and rigidity, that result in a conglomerate of uniform setae. However, the details of these setae can only be observed under SEM. Some spiders have several setae covering the lateral faces of femur IV, which can be mistaken as the femoral scopula. Peraf an et al. (2016), in their phylogenetic hypothesis for some Theraphosinae genera, included the femoral scopula in the morphological matrix as e.g. present in Cyrtopholis. Our ultrastructural analysis of specimens of Cyrtopholis revealed the presence of several thin and long setae, similar to those on other body parts, such as abdomen, carapace and legs, and therefore we do not considered them as femoral scopula. Before our work, the stridulating setae were considered as a unique structure, without variations. Our results showed that there are different stridulating setae that can implicate in taxonomics studies, for example, some stridulating seta types that are shared among close related genera, such as the claviform setae on trochanters and coxae in Acanthoscurria, Cyrtopholis and Phormictopus; the plumose setae on coxae in Lasiodora, Nhandu, Proshapalopus, Pterinopelma and Vitalius. However, the high diversity of setae described herein and by Galleti-Lima & Guadanucci (2018) combined with the ambiguous Theraphosinae relationship shampers any further discussion on stridulating setae evolution within the subfamily and Theraphosidae.
68
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
Fig. 59e61. 59. Eupalaestrus spinosissimus Mello-Leit~ao, 1923, female (arrow showing the spider scraping the femoral scopulae of leg IV against the abdomen). 60. Pterinopelma sazimai, female (arrows showing the femoral sopulae of legs I and IV). 61. Femoral scopulae of leg IV in the genus Vitalius. Images: A. Galleti-Lima & J.P.L. Guadanucci.
Acknowledgements Financial support for this study was obtained from CAPES, CNPq (process 479377/2012-0; process 168493/2017-9) and FAPESP (process 2017/11985-9). Thanks to the Department of Zoology and the Laboratory of Electronic Microscopy (Department of Biology), ~o Paulo State University (UNESP-Rio Institute of Biosciences of Sa ^nika Iamonte and Claro), and the technical support from Mo ^nio Yabuki. We also thank the curators for specimen loan, and Anto also Jorge Mendonza and David Ortiz for providing material. We are thankful to Stuart Longhorn, Rafael Indicatti, Marcio Bolfarini, Sylvia Lucas and Facundo Labarque for suggestions on an early draft of the manuscript, and also to Denis Pedroso for kindly sharing his spider body drawings. References Bertani, R., 2001. Revision, cladistic analysis, and zoogeography of Vitalius, Nhandu, and Proshapalopus; with notes on other Theraphosinae genera (Aranae, Theraphosidae). Arquivos de Zoologia 36 (3), 265e356. https://doi.org/10.11606/ issn.2176-7793.v36i3p265-356. Bertani, R., Leal, F., 2016. A new species of Pterinopelma (Araneae: Theraphosidae) from the highlands of the state of Minas Gerais, Brazil and description of the male of P. sazimai. Zoologia (Curitiba) 33 (2), 1e9. https://doi.org/10.1590/ S1984-4689zool-20150190. Bertani, R., Fukushima, C.S., Silva Jr., P.I., 2008. Two new species of Pamphobeteus Pocock 1901 (Araneae: Mygalomorphae: Theraphosidae) from Brazil, with a new type of stridulatory organ. Zootaxa 1826, 45e58. https://doi.org/10.11646/ zootaxa.1826.1.3. ^nero Grammostola Bücherl, W., 1951. Estudos sobre a biologia e a sistem atica do ge Simon, 1892, vol. 1. Monografias, Instituto Butantan, pp. 1e203. rez-Miles, F., 2011. The species of Grammostola (AraFerretti, N., Pompozzi, G., Pe neae: Theraphosidae) from Central Argentina: taxonomy, distribution, and surface ultrastructure of coxal setae. Zootaxa 2828, 1e18. https://doi.org/10. 11646/zootaxa.2828.1.1. Gabriel, R., 2014. A new genus and species of theraphosid spider from El Salvador (Araneae: Theraphosidae). Br. Tarantula Soc. J. 29 (3), 146e153. Galleti-Lima, A., Guadanucci, J.P.L., 2018. Morphology of setae on coxae and trochanters of Theraphosinae spiders (Mygalomorphae; Theraphosidae). J. Arachnol. 46 (2), 214e225. https://doi.org/10.1636/JoA-S-17-021.1. n al conocimiento de TherGerschman De, P.B.S., Schialelli, R.D., 1966. Contribucio rias do aphosa leblondi (Latreille), 1804 (Aranea: Theraphosidae), vol. 33. Memo Instituto Butantan, pp. 667e674.
mission de sons chez les Arane ides. Annee Biol. 2, Legendre, R., 1963. L’audition et l’e 371e390. Marroquin, J.I.M., 2014. Taxonomic revision of Hemirrhagus Simon, 1903 (Araneae: Theraphosidae, Theraphosinae), with description of five new species from Mexico. Zool. J. Linn. Soc. 170, 634e689. https://doi.org/10.1111/zoj.12112. Marshall, S.D., Thoms, E.M., Uetz, G.W., 1995. Setal entanglement: an undescribed method of stridulation by a neotropical tarantula (Araneae: Theraphosidae). J. Zool. 235, 587e595. https://doi.org/10.1111/j.1469-7998.1995.tb01770.x. Mendoza, J.I., Francke, O.F., 2018. Five new cave-dwelling species of Hemirrhagus Simon 1903 (Araneae, Theraphosidae, Theraphosinae), with notes on the generic distribution and novel morphological features. Zootaxa 4407 (4), 451e482. https://doi.org/10.11646/zootaxa.4407.4.1. Ortiz, D., Bertani, R., 2005. A new species in the spider genus Phormictopus (Therrica de Aracnología 11, aphosidae: Theraphosinae) from Cuba. Revista Ibe 29e36. Peraf an, C., Cifuentes, Y., Estrada-Gomez, S., 2015. Aguapanela, a new tarantula genus from the Colombian Andes (Araneae, Theraphosidae). Zootaxa 4033 (4), 529e542. https://doi.org/10.11646/zootaxa.4033.4.4. rrez, M., Pe rez-Miles, F., 2016. Kankuamo, a new therPeraf an, C., Galvis, W., Gutie aphosid genus from Colombia (Araneae, Mygalomorphae), with a new type of urticating setae and divergent male genitalia. ZooKeys 601, 89e109. https://doi. org/10.3897/zookeys.601.7704. rez-Miles, F., Lucas, S.M., Silva, P.I., Bertani, R., 1996. Systematic revision and Pe cladistic analysis of Theraphosinae (Araneae: Theraphosidae). Mygalomorph 1, 33e68. rez-Miles, F., Oca, L.M.D., Postiglioni, R., Costa, F.G., 2005. The stridulatory setae of Pe Acanthoscurria suina (Araneae, Theraphosidae) and their possible role in sexual rie Zool. 95 (4), communication: an experimental approach. Iheringia. Se 365e371. https://doi.org/10.1590/S0073-47212005000400004. Pocock, R.I., 1895. Musical boxes in spiders. Nat. Sci. 6, 44e50. Ramírez, M.J., 2014. The morphology and phylogeny of Dionychan spiders (Araneae: Araneomorphae). Bull. Am. Mus. Nat. Hist. 390, 1e374. Raven, R.J., 1985. The spider infraorder Mygalomorphae (Araneae): cladistics and systematics. Bull. Am. Mus. Nat. Hist. 182, 1e180. Rudloff, J.P., Weinmann, D., 2010. A new giant tarantula from Guyana. Arthropoda Scientia 1, 21e40. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., Cardona, A., 2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9 (7), 676e682. https:// doi.org/10.1038/nmeth.2019. Schmidt, G., 2003. Die Vogelspinnen: Eine weltweite Übersicht. Hohenwarsleben. Neue Brehm-Bücherei 641, 1e383. Smith, A.M., 1990. Baboon Spiders. Tarantulas of Africa and the Middle East, vol. 1. Fitzgerald Publishing London, p. 142. Uetz, G.W., Stratton, G.E., 1982. Acoustic Communication and Reproductive Isolation in Spiders. Spider Communication: Mechanisms and Ecological Significance. Princeton University Press, Princeton, New Jersey, pp. 123e159.
Contents lists available at ScienceDirect
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Research paper
Comparative morphology of stridulating setae of Theraphosinae (Araneae: Theraphosidae) Paulo Leite Guadanucci Arthur Galleti-Lima*, Jose ~o Paulo State University (UNESP e Rio Claro), Rio Claro, Sa ~o Paulo, Brazil Department of Zoology, Institute of Biosciences, Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 March 2019 Received in revised form 25 June 2019 Accepted 30 August 2019 Available online 7 September 2019
Theraphosid spiders are repleted with cuticular structures that evolved into distinct morphologies and developed different functions. The stridulatory apparatus present in the subfamily Theraphosinae is composed by a group of setae and opposed organs (ridged surface) that emit sound through friction. Only sixteen genera of Theraphosinae spiders were reported with stridulatory setae: Acanthoscurria, Aguapanela, Brachypelma, Citharacanthus, Cyrtopholis, Grammostola, Hemirrhagus, Lasiodora, Longilyra, Nhandu, Theraphosa, Pamphobeteus, Phormictopus, Proshapalopus, Pterinopelma and Vitalius. Besides these stridulating setae, other cuticular structures have been traditionally used in Theraphosinae taxonomy: the plumose scopula on the lateral faces of femora I and IV. However, the ultrastructure of these setae and scopulae was never studied in detail. Here, we analyzed the diversity of cuticular structures on coxae, trochanters and femora of Theraphosinae spiders using SEM. Beside the description of plumose and claviform stridulating setae on femora, we established five distinct variations of stridulatory setae found on coxae and trochanters and report their presence for the genus Megaphobema, Crassicrus and Cotztetlana for the first time. © 2019 Elsevier GmbH. All rights reserved.
Corresponding Editor: P. Michalik Keywords: Tarantula Ultrastructure Stridulation SEM
1. Introduction The spider family Theraphosidae is currently divided in eleven subfamilies with the neotropical subfamily Theraphosinae as the most diverse group. The presence of setae, or groups of setae, forming structures named lyra, stridulatory apparatus and scopulae, mainly on basal articles of legs and palps, such as coxae and trochanters is very common within Theraphosidae (Raven 1985; Smith 1990). Many group diagnoses are based on the possession of these distinct setae, and some of these spiders are notorious for producing sound using some body setae (Uetz & Stratton 1982; Marshal et al. 1995). This phenomenon known as stridulation (Pocock 1895) is the result of the friction of these structures, which are often located on coxae and trochanters of legs and palps (Marshall et al. 1995). Some authors have suggested sexual or defensive roles for such stridulation. (Legendre 1963; Uetz & Stratton 1982; Marshall et al. 1995). According to Uetz & Stratton (1982), the stridulation occurs by the scratching of structures located on opposite faces, producing
* Corresponding author. E-mail addresses: [email protected] [email protected] (J.P.L. Guadanucci). https://doi.org/10.1016/j.jcz.2019.08.010 0044-5231/© 2019 Elsevier GmbH. All rights reserved.
(A.
Galleti-Lima),
vibrations and, in some cases, audible sounds for humans. The knowledge about occurrence and morphology of stridulating setae in Theraphosinae is scarce and limited to taxonomic studies without an inter-generic comparative approach. The majority of the taxonomic literature refers to the occurrence of these structures, however, SEM data are present in just a few of them. To date, the following Theraphosinae genera were reported with stridulating setae: Acanthoscurria Ausserer, 1871; Aguapanela n & Cifuentes, 2015; Brachypelma Simon, 1891; Cyrtopholis Perafa Simon, 1892; Citharacanthus Pocock, 1901; Grammostola Simon, 1892; Hemirrhagus Simon, 1903; Lasiodora C. L. Koch, 1850; Longilyra Gabriel, 2014; Nhandu Lucas, 1983; Pamphobeteus Pocock, ~o, 1901; Phormictopus Pocock, 1901; Proshapalopus Mello-Leita 1923; Pterinopelma Pocock, 1901; Theraphosa Therell, 1870 and Vitalius Lucas, Silva & Bertani, 1993. However, not all of these representatives had their coxae and/or trochanter examined under the SEM, which is essential for a proper recognition of such setae. Besides stridulating setae on coxae and trochanter, Theraphosinae spiders are replete with other setae types of taxonomic relevance. According to Raven (1985), the presence of scopulae on the retrolateral face of femur IV is synapomorphic for the subfamily Theraphosinae. The occurrence of these scopula e was also used as a rez-Miles et al. (1996) and Peraf character by Pe an et al. (2016) in
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
their phylogenetic hypotheses of Theraphosinae intrarelationships. However, contrary to Raven (1985), it was shown that many Theraphosinae representatives lack the femoral scopula. According to Peraf an et al. (2016), the following genera are characterized with the presence of femoral scopula: Acanthoscurria, Cyrtopholis, Eupalaestrus Pocock, 1901, Lasiodora, Megaphobema Pocock, 1901, Metriopelma Becker, 1878, Nhandu, Pamphobeteus, Phormictopus, Schizopelma F. O. Pickard-Cambridge, 1897, Sericopelma Ausserer, 1875, Theraphosa, Vitalius e Xenesthis Simon, 1891. In this study, we performed a comparative ultrastructural analysis of stridulatory setae of Theraphosinae representatives. Furthermore, we present an inventory of these setae and propose a standard terminology for Theraphosinae taxonomy complementing the descriptions in Galleti-Lima & Guadanucci (2018). 2. Material and methods The material examined belongs to the following scientific col~o Paulo, Brasil (IBSP) e A. Brescovit; lections: Instituto Butantan, Sa ^ncias Naturais, Fundaça ~o Zoobot^ Museu de Cie anica do Rio Grande n Nacional de do Sul, Porto Alegre, Brasil (MCN) e R. Ott; Coleccio cnidos UNAM, Me xico DF, Me xico (CNAN) e O. F Ballve ; Centro Ara ^ncias Exatas e da Natureza, Universidade Federal da Paraíba, de Cie ~o Pessoa, Brazil (CCEN) e M. B. da Silva; Coleça ~o Aracnolo gica Joa Diamantina, Rio Claro, Brazil (CAD) e J. P. L Guadanucci; Personal n Garcia. Collection Alayo Material examined (geographic coordinates between parentheses indicate the location of the municipality, obtained from Google earth): ~o, 1923: 1 _, 1 \ (CAD Acanthoscurria gomesiana Mello-Leita 472). Ami sp.: 1 _, (MPEG 02-220), 1 \, (MPEG 02-088). Aphonopelma anitahoffmannae Locht et al., 2005: 1 _, 1 \, (CNAN 3047). Brachypelma smithi F. O. Pickard-Cambridge, 1897: 1 _, Exoesqueleton. Bumba sp.: 1 _, (IBSP 151865). Catanduba flavohirta Simon, 1889: 1 _, (IBSP 126901). Catanduba sp.: 1 _, (CAD). Cotztetlana sp.: 1 \, Exoesqueleton. Crassicrus sp.: 1 \, Exoesqueleton. rez-Miles, 1998: 1 _, (CAD), 1 \, (CAD). Cyriocosmus chicoi Pe Cyrtopholis sp.: 1 _, (IBSP), 1 \, (IBSP). Davus sp.: 1 _, (CNAN 3515). Euathlus sp.: 1 _, (IBSP 3817-A). Eupalaestrus sp.: 1 _, (IBSP 4200). Grammostola sp.: 1 _, (IBSP 2427), 1 \, (IBSP 2427). Hapalopus rez-Miles, 1998.: 1 _, (MPEG 019077), 1 \, (MPEG butantan Pe rez-Miles & Locht, 2003: 1 _, 019081). Hemirrhagus papalotl Pe (CNAN). Hemirrhagus sp.: 1 \, (CNAN-Ar. 010269). Hemirrhagus billsteelei Mendoza & Francke, 2018: 1 _, (CNANAr. 010274). Hemirrhagus kalebi Mendoza & Francke, 2018: 1 _, (CNAN). Homoeomma montanum Mello-Leit~ ao, 1923: 1 _, (IBSP 4685). Kochiana sp.: 1 _, (IBSP 11716). Lasiodora klugi C. L. Koch, 1841.: 1 _, (CCEN. 181). Magulla obesa Simon, 1892: 1 _, (IBSP 8367), 1 \, rez-Miles, Miglio & Bonaldo, 2006: (CAD). Megaphobema teceae Pe 1 _, (MPEG 005233). Nhandu cerradensis Bertani, 2001: 1 _, (IBSP 11847); 1 \, (IBSP 13971). Pamphobeteus augusti Simon, 1889: 1 _, n Garcia. Plesiopelma sp.: 1 \, ExoesPersonal Collection Alayo queleton. Phormictopus cautus Ausserer, 1875: 1 _, (IES, CZAAC ~o, 1929: 1 _, 3.4244). Proshapalopus multicuspidatus Mello-Leita (CAD 094). Pterinopelma felipeleitei Bertani & Leal, 2016: 1 _, (CAD 441), 1 \, (CAD 584). Pterinopelma sazimai Bertani, Nagahama & Fukushima, 2011: 1 _, (CAD), 1 \ (CAD). Pterinopelma vitiosum Keyserling, 1891: 1 _, (MCN 22145), 1 \, (MCN 22102). Theraphosa blondi Latreille, 1804: 1 _ (MPEG ARA 000176), 1 \, (MPEG 007558). Tmesiphantes sp.: 1 _, (CAD). Vitalius sp.: 1 _, (CAD). 2.1. Scanning Electron Microscopy (SEM) Coxae, trochanters and femora were dissected and manually cleaned with brush. Then the material was ultrasonic cleaned,
59
using a water and detergent solution. After ultrasonic cleaning, the material underwent dehydration in graded ethanol (70%, 80%, 90% and absolute ethanol e 60 min in each concentration) and were critical-point dried. The material was glued to stubs with doublesided copper tapes, sputter-coated with gold and examined in a Hitachi TM- 1000 Scanning Electronic Microcopy, at the Electronic Microscopy Laboratory of the Institute of Biosciences of Sao Paulo ~o Paulo, Brazil. State University, campus Rio Claro, Sa 2.2. Seta types and morphometry All setae present on the opposite faces of coxae, trochanters and femora with distinct morphological features different from the surrounding regular setae were investigated. We considered regular setae as those present also on other body parts, such as carapace, sternum, abdomen, spinnerets and other leg articles. The lengths of setae were measured with the software ImageJ Fiji 32-bit (Schindelin et al. 2012). Whenever possible, five setae were measured per specimen. Pictures were edited for brightness/ contrast corrections and grouped in plates with Photoshop CS6. 3. Results In order to apply a standardized description for stridulatory rez-Miles et al. (2005) and Galleti-Lima & setae, we followed Pe Guadanucci (2018), who established morphological patterns and terminology for this type of setae. Unfortunately, we did not have specimens of the genera Longilyra and Citharacanthus, and therefore used original descriptions for our analysis. Table 1 shows the distribution, morphology and morphometric data of stridulatory setae examined in representatives of Theraphosinae. Those specimens included in the list of material examined but not present in any table, figure or through the text henceforth, do not possess any type of stridulating setae. 3.1. Pilose stridulating setae These setae are only present in Hemirrhagus sp. and are characterized by having a smooth base with modestly long barbs extending from the shaft region to the apex. They differ from the plumose stridulating setae (Peraf an et al. 2015), by the lack of grooves at the apex surface (Figs. 9e12). The pilose stridulating setae arrangement can be seen in Fig. 40. In addition, we found two variations, thin (Figs. 9e10), restricted to coxae, and thick (Figs. 11e12), restricted to trochanters. 3.2. Sectioned stridulating setae The sectioned stridulating setae are characterized by the robust appearance and the smooth base that extends up to approximately the mid length, where small barbs are arranged in parallel lines and extend to the apex, giving it the appearance of longitudinal sectioned regions marked by conspicuous longitudinal furrows (Figs. 1e2). This seta type was observed in Aphonopelma annitahoffmannae, H. billsteelei and H. kalebi. The arrangement of these setae is shown in Figs. 31, 34, 41 and 42. 3.3. Spatuliform stridulating setae These setae are characterized by having a smooth base and a mid region with a membrane-like projection covered with small short and thin beards, extending to the apex (Figs. 13e14). They were found in H. billsteelei and H. kalebi and their arrangement can be observed in Figs. 41 and 42.
60
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
Table 1 Distribution, morphology and morphometry of stridulatory setae of Theraphosinae genera. Measurements are in micrometers (mm); SD ¼ standard deviation; n ¼ sample size, () ¼ not examined. The spiniform setae of the genus Pamphobeteus is common in other Theraphosidae spiders. Stridulating setae type
Genera
Figures setae
Sectioned
Aphonopelma
1e2, 31 and 32
Sectioned Sectioned
Citharacanthus Hemirrhagus billsteelei
Schmidt (2003): fig. 190, 191, 293 and 294; Fig. 34 Figs. 1e2 and 53
Sectioned
Hemirrhagus kalebi
Figs. 1e2 and 54
Spiniform
Cotztetlana
Figs. 3 and 35
Spiniform
Theraphosa
Galleti-Lima & Guadanucci (2018): fig. 14; Fig. 57
Plumose
Crassicrus
Figs. 4 and 36; Seta detail in Galleti-Lima & Guadanucci (2018): figs. 19e22
Plumose
Megaphobema
Figs. 5 and 45
Plumose
Acanthoscurria
Galleti-Lima & Guadanucci (2018): 11, 12 and 13; Fig. 29
Plumose Plumose
Aguapanela Lasiodora
Peraf an et al. (2015): figs 1e12; Fig. 30 Galleti-Lima & Guadanucci (2018): fig. 8; Fig. 43
Plumose
Nhandu
Galleti-Lima & Guadanucci (2018): fig. 9; Figs. 46 and 47
Plumose
Proshapalopus
Galleti-Lima & Guadanucci (2018): fig. 6; Fig. 50
Plumose
Pterinopelma
Galleti-Lima & Guadanucci (2018): fig. 7; Figs. 51e56
Plumose
Vitalius
Galleti-Lima & Guadanucci (2018): fig. 10; Fig. 58
Claviform Claviform
Phormictopus Acanthoscurria
Figs. 6 and 49; Seta detail in Galleti-Lima & Guadanucci (2018): figs. 15e18 Galleti-Lima & Guadanucci (2018): figs. 1; and 29
Claviform
Brachypelma
Galleti-Lima & Guadanucci (2018): fig. 3; Fig. 33
Claviform
Cyrtopholis
Galleti-Lima & Guadanucci (2018): fig. 2; Fig. 37
Claviform
Theraphosa
Galleti-Lima & Guadanucci (2018): fig. 4; Fig. 57
Rough
Grammostola
Figs. 7e8 and 39
Pilose thin
Hemirrhagus sp.
Figs. 9e10 and 40
Pilose thick
Hemirrhagus sp.
Figs. 11e12 and 40
Spatuliform
Hemirrhagus billsteelei
Figs. 13e14 and 41
Spatuliform Spiniform (common variation) Velvet
Hemirrhagus kalebi Pamphobeteus
Figs. 13e14 and 42 Figs. 15e16 and 48
Lasiodora
Galleti-Lima & Guadanucci (2018): figs. 5 and 8; Fig. 43
Liriform
Longilyra
Gabriel (2014): figs 1e3; Fig. 44
Average Length e mm (±SD) _
\
271,4 (n ¼ 1) e 503.0 (±503.0) (n ¼ 3) 327.6 (±19.2) (n ¼ 3) e
e
938.7 (±178.2) (n ¼ 3) e 517.4 (±99.0) (n ¼ 4) 584.8 (±79.4) (n ¼ 4) e 817.3 (±130.7) (n ¼ 5) 413.2 (±60.1) (n ¼ 3) 375.2 (±81.3) (n ¼ 5) 610.8 (±147.8) (n ¼ 3) 616.1 (±67.5) (n ¼ 4) 1147.1 (n ¼ 1) 594.7 (±203.1) (n ¼ 5) 577.5 (±268.2) (n ¼ 5) 425.9 (±43.1) (n ¼ 2) 420.9 (n ¼ 1) 682.3 (±140.3) (n ¼ 3) 368.7 (±182.5) (n ¼ 4) 446.1 (±181.0) (n ¼ 2) 627.9 (±151.5) (n ¼ 5) 181.0 (n ¼ 1) e 1056.2 (±150.9) (n ¼ 3) e
Fig. 1e2. Sectioned stridulating setae of Aphonopelma annitahoffmannae. 1. Overview. 2. Apex. Images: A. Galleti-Lima.
e e e 433.3 (±95.3) (n ¼ 4) 668.3 (±296.2) (n ¼ 5) 529.7 (±60.1) (n ¼ 3) e 538.1 (±130.2) (n ¼ 4) e e 566.5 (±70.0) (n ¼ 5) e 480.178 (±63.7) (n ¼ 2) e e 259.2 (±98.2) (n ¼ 5) e 316.7 (±37.9) (n ¼ 4) 1039.4 (±182; 6) (n ¼ 2) 685.8 (±244.6) (n ¼ 4) e e e e e e e
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
61
Fig. 3e6. Types of stridulating setae. 3. Spiniform stridulating setae of Cotztetlana. 4. Plumose stridulating setae of Crassicrus (arrows showing the stridulating setae). 5. Plumose stridulating setae of Megaphobema. 6. Claviform stridulating setae of Phormictopus. Images: A. Galleti-Lima.
Fig. 7e8. Rough stridulating setae of Grammostola sp. 7. Overview. 8. Apex. Images: A. Galleti-Lima.
Fig. 9e10. Pilose stridulating setae (thin variation) of Hemirrhagus sp. 9. Overview. 10. Apex. Images: A. Galleti-Lima.
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Fig. 11e12. Pilose stridulating setae (thick variation) of Hemirrhagus sp. 11. Overview. 12. Apex. Images: A. Galleti-Lima.
Fig. 13e14. Spatuliform stridulating setae of Hemirrhagus billsteelei. 13. Overview. 14. Apex. Images: A. Galleti-Lima.
Fig. 15e16. Tactile spiniform setae of Pamphobeteus augusti. 15. Overview (arrows showing the tactile setae). 16. Apex. Images: A. Galleti-Lima.
3.4. Rough stridulating setae The rough stridulating setae type is reported for Grammostola, being characterized by a smooth base and a middle region completely covered by short barbs that extend to the apex (Figs. 7e8). The arrangement of these setae in coxae and trochanters are shown in Fig. 39. 3.5. Liriform stridulating setae The description of this type of seta is based on data from Gabriel (2014), as we did not have material available to examine. Their images show parallel disposed setae, which have a feather-like aspect and swollen apex, present in spiders of the genus Longilyra Gabriel, 2014 on the prolateral trochanter of leg I (Fig. 44). The number of setae ranges from 11 to 16 (Gabriel 2014). The terminology for these setae refers to the original description of the genus
(Gabriel 2014), as the term lyra was used by the author to describe the stridulating setae, which are disposed in parallel. Distinct types of stridulating setae may occur on the same individual, as reported by Galleti-Lima & Guadanucci (2018) for the genera Acanthoscurria, Lasiodora and Theraphosa. In the present work, we observed the same organization for H. billsteelei, with spatuliform and sectioned types. Regarding the scopula on femora IV, our ultrastructural analysis revealed plumose stridulating setae on the retrolateral side in representatives of the genera Acanthoscurria (Fig. 29), Eupalaestrus (Fig. 38), Lasiodora (Fig. 43), Megaphobema (Fig. 45), Nhandu (Figs. 46e47), Pamphobeteus (Fig. 48), Phormictopus (Fig. 49), Proshapalopus (Fig. 50), Pterinopelma (Figs. 51e56) and Vitalius (Fig. 58). The genera Acanthoscurria, Eupalaestrus, Lasiodora, Nhandu and Vitalius also have femoral scopula (plumose stridulating setae) on prolateral femur I, distinguishing them from the remaining taxa that have femoral scopula on femur IV only. All
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63
Fig. 17e24. Stridulating setae of femoral scopula. 17. Plumose stridulating setae of the femora I and IV of Acanthoscurria (arrows showing the stridulating setae). 18. Claviform stridulating setae of the palpal femur, legs I, II, III and IV of Brachypelma. 19. Plumose stridulating setae of femora I and IV of Eupalaestrus. 20. Plumose stridulating setae of femora I, II, III and IV of Lasiodora. 21. Plumose stridulating setae of femora I and IV of Megaphobema (arrows showing the stridulating setae). 22. Plumose stridulating setae of femora I and IV of Nhandu. 23. Plumose stridulating setae of the femur IV of Pamphobeteus. 24. Plumose stridulating setae of the femur IV of Phormictopus. Images: A. Galleti-Lima.
investigated specimen are characterized by a less dense femoral scopula on leg I compare to that on femur IV. The genera Brachypelma and Theraphosa also present femoral scopula on the palps (retrolateral) and leg femora I-IV (see different distributions in Figs. 33 and 57), but these are composed by claviform stridulating setae. Genera with stridulating setae on its femora are listed in Table 2, with seta type, occurrence and morphometric data. Figs. 29e58 show the distribution of femoral scopula, as well as the stridulating apparatus on coxae and trochanters.
4. Discussion All stridulatory setae examined herein, and also those described in Galleti-Lima & Guadanucci (2018) among numerous other studies (e.g. Bücherl 1951; Gerschman & Schiapelli 1966; Marshall rez-Miles et al. 2005; Rudloff & et al. 1995; Bertani 2001; Pe Weinmann 2010), share the following features: (1)- restricted to opposite faces (coxae, trochanters and femura), instead of spreading through the body, or opposed to another structure (e.g.
64
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Fig. 25e28. Stridulating setae of the femoral scopula. 25. Plumose stridulating setae of the femur IV of Pterinopelma (arrows showing the stridulating setae). 26. Plumose stridulating setae of femur IV Proshapalopus. 27. Claviform stridulating setae legs I, II, III and IV femora of Theraphosa. 28. Plumose stridulating setae of femora I and IV of Vitalius (arrows showing the stridulating setae). Images: A. Galleti-Lima.
other setae, ridges) to warrant friction for producing vibration; (2) rigidness, resisting attempts to bent with a needle or tweezers; (3) surface with rough texture, with barbs or ridges. All these features seem to be a requirement to produce vibration, which in some cases are audible hissing sound for humans. Many other regular body setae also have barbs, such as the tactile setae, which in Theraphosidae are characterized by the insertion on a socket and very short barbs, or the scale-like setae, which are very distinct from other setae as their base strongly bent (Ramirez 2014). These types of setae, can participate of the stridulation, but apparently not rez-Miles et al. 2005; are necessarily obligatory in the process (Pe Ferretti et al. 2011). Moreover, groups of stridulatory setae are very distinct from any other setae around them. Although in some cases they are hard to identify under the stereomicroscope, they are easy to distinguish under the SEM. 4.1. Plumose setae Galleti-Lima & Guadanucci (2018) highlighted the importance of the examination of the seta ultrastructure in order to characterize and identify the morphological patterns. Ortiz & Bertani (2005) used the term “plumose seta” to describe the stridulatory apparatus of Phormictopus, examined under the stereomicroscope. Galleti-Lima & Guadanucci (2018) did not have specimens of Phormictopus available for examination and assumed that this seta should be the claviform setae, based on the close evolutionary affinities with Acanthoscurria and Cyrtopholis. The data presented here confirms the claviform condition of Phormictopus stridulating setae. According to Galleti-Lima & Guadanucci (2018), the claviform stridulating setae found in the genus Phormictopus can be considered as an additional generic diagnostic feature, including Brachyn et al. pelma, Acanthoscurria, Cyrtopholis and Theraphosa. Perafa (2015), reported tufts of stridulating setae for spiders of the genus Aguapanela. This new variation of stridulating setae, was named stridulating plumose setae and were found on prolateral face of coxae, trochanters and femora of legs I and II. Galleti-Lima &
Guadanucci (2018) found the same type of setae when investigating these tufts in representatives of Acanthoscurria, Proshapalopus, Pterinopelma, Lasiodora, Nhandu and Vitalius. According to Marroquin (2014) and Mendoza & Francke (2018), some species of the genus Hemirrhagus possess a variety of stridulating setae on lateral faces of trochanters and coxae of legs and palp: Hemirrhagus akheronteus Mendoza & Francke, 2018 (feathery), H. billsteelei (claviform and spiniform), Hemirrhagus rez-Miles & Locht, 2003 (plumose), Hemirrhagus diablo chilango Pe Mendoza & Francke, 2018 (plumose), Hemirrhagus franckei Mendoza, 2014 (spiniform and plumose), H. kalebi (parallel and plumose claviform), Hemirrhagus nahuanus Gertsch, 1982 (spiniform and plumose), Hemirrhagus perezmilesi García-Villafuerte & Locht, 2010 (spiniform and claviform), Hemirrhagus pernix Ausserer, 1875 (spiniform and plumose), Hemirrhagus reddelli Gertsch, 1973 (spiniform, plumose and stridulating setae on femur) and Hemirrhagus sprousei Mendoza & Francke, 2018 (plumose and spiniform). These Hemirrhagus species were not examined under the SEM and the setal names given above are the ones originally proposed by Marroquin (2014) and Mendoza & Francke (2018) not matching with the terminology adopted here and in Galleti-Lima & Guadanucci (2018). In our study, we could only investigate the ultrastructure for three species of Hemirrhagus, which possess the following types of stridulating setae using terminology in GalletiLima & Guadanucci (2018): Hemirrhagus sp. with pilose setae (thin and thick variants); H. billsteelei and H. kalebi with spatuliform setae. The number of stridulating setae found in only three species of Hemirrhagus indicates that the genus possesses a potential for the occurrence of other variations (including new variations), which justifies precise analysis of legs articles under SEM. 4.2. Sectioned stridulating setae Schmidt (2003) reported sectioned stridulating setae for the genus Citharacanthus, on trochanters of palps (retrolateral) and legs I (prolateral) (Fig. 34). Considering the close phylogenetic
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65
Fig. 29e44. Occurrence of stridulating setae on coxae, trochanters and femora. 29. Acanthoscurria gomesiana. 30. Aguapanela sp. 31-32. Aphonopelma annitahoffmanae. 33. Brachypelma smithi. 34. Citharacanthus sp. 35. Cotztetlana sp. 36. Crassicrus sp. 37. Cyrtopholis sp. 38. Eupalaestrus sp. 39. Grammostola sp. 40. Hemirrhagus sp. 41. Hemirrhagus billsteelei. 42. Hemirrhagus kalebi. 43. Lasiodora klugi. 44. Longilyra sp. Drawing credit: Denis Rafael Pedroso.
n et al., 2016), we expected relationship with Aphonopelma (Perafa that this type of stridulating setae is similar in both genera. However, representatives of the genus Citharacanthus have not been examined under SEM and this hypothesis still needs to be tested.
4.3. Spiniform setae Many authors have used the term “spiniform” to refer to putative stridulating setae, i. e. distinct setae positioned on opposite
66
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
Fig. 45e58. Occurrence of stridulating setae on coxae, trochanters and femora. 45. Megaphobema teceae. 46-47. Nhandu cerradensis. 48. Pamphobeteus augusti. 49. Phormictopus cautus. 50. Proshapalopus multicuspidatus. 51-52. Pterinopelma felipeleitei. 53-54. Pterinopelma sazimai. 55-56. Pterinopelma vitiosum. 57. Theraphosa blondi. 58. Vitalius sp. Drawing credit: Denis Rafael Pedroso.
faces on legs and/or palp articles. In the original description of Pamphobeteus crassifemur and P. grandis, Bertani et al. (2008 e see Figs. 9e12) reported the presence of “spiniform setae” on lateral faces of coxae and trochanters. Bertani et al. (2008) suggested that stridulation in P. crassifemur takes place while the spider sheds urticating setae, when lifting its hind legs. However, a different possibility of a stridulating site in Pamphobeteus could be at femur
IV (plumose retrolateral scopula) (Fig. 48), which might scrap against the anterior lateral faces of abdomen. Although this process of stridulation has never been reported before, our morphological analysis revealed that the femoral scopula is composed of the same type of stridulating setae (plumose or claviform) on coxae and trochanters resembling a potential stridulating apparatus. We found a distinct group of long setae in P. augusti, similar to tactile
A. Galleti-Lima, J.P.L. Guadanucci / Zoologischer Anzeiger 283 (2019) 58e68
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Table 2 Femoral scopulae on Theraphosinae genera: type, occurrence and morphometrics. Stridulating setae
Genera
Femur
Average Length (mm)
Figures
Claviform
Brachypelma
All legs þ palp
Figs. 18 and 33
Claviform
Theraphosa
All legs þ palp
Plumose
Acanthoscurria
I and IV
Plumose Plumose
Eupalaestrus Lasiodora
I and IV All
Plumose Plumose
Megaphobema Nhandu
IV I and IV
Plumose
Pamphobeteus
IV
Plumose
Phormictopus
IV
Plumose
Pterinopelma
Plumose
Proshapalopus
I and IV (P. sazimai) IV (remaining spp) IV
Plumose
Vitalius
I and IV
440.0 (n ¼ 1) 735.3 (±122.8) (n ¼ 4) 695.4 (±131.1) (n ¼ 2) 390.0 (n ¼ 1) 908.2 (±486.3) (n ¼ 4) 1113.7 (n ¼ 1) 731.7 (±117.5) (n ¼ 2) 624.0 (±112.4) (n ¼ 5) 345.8 (n ¼ 1) 559.6 (n ¼ 1) 335.3 (n ¼ 1) 657.1 (±313.2) (n ¼ 5)
setae present on legs as they share the same ultrastructure and the insertion in a socket (Figs. 15e16). We are still far from knowing the whole morphological variants and the process of stridulating in Pamphobeteus. Further detailed micrographies, with the inclusion of a greater diversity of Pamphobeteus species, are still needed to reach a standard terminology. Behavioral studies focused on stridulation may clarify the process of stridulation in Pamphobeteus species. Ferreti et al. (2011: Figs. 4e7, 10, 12) also used the term “spiniform” referring to setae, which were found interspersed with plumose setae, located on opposite faces of coxae and maxillae in Grammostola. They recognized two variants of the same type (short and long) and presented ultrastructural data showing their similarities with regular leg spines: unbarbed, with ridged surface. rez-Miles et al. (2005), the According to Ferretti et al. (2011) and Pe spiniform setae can also participate in the stridulating process, since they are rigid and with a rough surface. Galleti-Lima & Guadanucci (2018: Figs. 14, 27e30) also reported the presence of “spiniform” setae in Theraphosa and Cotztetlana. Gerschamn & Schiapelli (1966) had also reported these in Theraphosa, and they used the term “black spines”. In fact, the spiniform setae in Theraphosa, Grammostola and Cotztetlana are very similar to leg spines and, until behavior studies are carried out, the role in stridulation is questionable. Concerning setal terminology, we promote the use of the term “spiniform” for these specific setae in Theraphosa, Grammostola and Cotztetlana. Those spiders belonging to genera that possess putative stridulating setae, that meet the three conditions for stridulating presented herein, and are notorious for their ability to produce audible sound (e. g. Lasiodora, Theraphosa) making them prime candidates for further investigations. 4.4. Setae on femoral scopula Regarding the femoral scopula across Theraphosinae, the distinct organizations depicted in Figs. 29e58 can be used as diagnostic characters for genera recognition. For example, the close relationship between P. sazimai with Vitalius and Nhandu as recovered in the phylogenetic hypothesis in Galleti-Lima &
Figs. 27 and 57 Figs. 17 and 29 Figs. 19 and 38 Figs. 20 and 43 Figs. 21 and 45 Figs. 22, 46 and 47 Figs. 23 and 48 Figs. 24 and 49 Figs. 25, 51e56 Figs. 26 and 50 Figs. 28 and 58
Guadanucci (2018) and also suggested by Bertani & Leal (2016), receives further support with the presence of femoral scopula on leg I (prolateral). The remaining Pterinopelma species (P. felipeleitei and P. vitiosum) lack femoral scopula on leg I. It might be possible to distinguish the plumose and claviform setae of femoral scopula from the other setae using a stereomicroscope (magnifications above 50). However, the identification of such setae can only reliable be done using SEM. This also explains that many described species lack information concerning the femoral scopula, and other stridulating setae on coxae and trochanter (e.g. Vitalius, Pterinopelma, Nhandu and Proshapalopus). Figs. 59 and 60 show a fe~o, 1923 and a female male Eupalaestrus spinosissimus Mello-Leita P. sazimai with conspicuous femoral scopula, which is depicted in detail (stereomicroscope) in Fig. 61. This femoral scopula can be easily observed, with the help of a stereomicroscope or not, because the setae that constitute this scopula have the same length and rigidity, that result in a conglomerate of uniform setae. However, the details of these setae can only be observed under SEM. Some spiders have several setae covering the lateral faces of femur IV, which can be mistaken as the femoral scopula. Peraf an et al. (2016), in their phylogenetic hypothesis for some Theraphosinae genera, included the femoral scopula in the morphological matrix as e.g. present in Cyrtopholis. Our ultrastructural analysis of specimens of Cyrtopholis revealed the presence of several thin and long setae, similar to those on other body parts, such as abdomen, carapace and legs, and therefore we do not considered them as femoral scopula. Before our work, the stridulating setae were considered as a unique structure, without variations. Our results showed that there are different stridulating setae that can implicate in taxonomics studies, for example, some stridulating seta types that are shared among close related genera, such as the claviform setae on trochanters and coxae in Acanthoscurria, Cyrtopholis and Phormictopus; the plumose setae on coxae in Lasiodora, Nhandu, Proshapalopus, Pterinopelma and Vitalius. However, the high diversity of setae described herein and by Galleti-Lima & Guadanucci (2018) combined with the ambiguous Theraphosinae relationship shampers any further discussion on stridulating setae evolution within the subfamily and Theraphosidae.
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Fig. 59e61. 59. Eupalaestrus spinosissimus Mello-Leit~ao, 1923, female (arrow showing the spider scraping the femoral scopulae of leg IV against the abdomen). 60. Pterinopelma sazimai, female (arrows showing the femoral sopulae of legs I and IV). 61. Femoral scopulae of leg IV in the genus Vitalius. Images: A. Galleti-Lima & J.P.L. Guadanucci.
Acknowledgements Financial support for this study was obtained from CAPES, CNPq (process 479377/2012-0; process 168493/2017-9) and FAPESP (process 2017/11985-9). Thanks to the Department of Zoology and the Laboratory of Electronic Microscopy (Department of Biology), ~o Paulo State University (UNESP-Rio Institute of Biosciences of Sa ^nika Iamonte and Claro), and the technical support from Mo ^nio Yabuki. We also thank the curators for specimen loan, and Anto also Jorge Mendonza and David Ortiz for providing material. We are thankful to Stuart Longhorn, Rafael Indicatti, Marcio Bolfarini, Sylvia Lucas and Facundo Labarque for suggestions on an early draft of the manuscript, and also to Denis Pedroso for kindly sharing his spider body drawings. References Bertani, R., 2001. Revision, cladistic analysis, and zoogeography of Vitalius, Nhandu, and Proshapalopus; with notes on other Theraphosinae genera (Aranae, Theraphosidae). Arquivos de Zoologia 36 (3), 265e356. https://doi.org/10.11606/ issn.2176-7793.v36i3p265-356. Bertani, R., Leal, F., 2016. A new species of Pterinopelma (Araneae: Theraphosidae) from the highlands of the state of Minas Gerais, Brazil and description of the male of P. sazimai. Zoologia (Curitiba) 33 (2), 1e9. https://doi.org/10.1590/ S1984-4689zool-20150190. Bertani, R., Fukushima, C.S., Silva Jr., P.I., 2008. Two new species of Pamphobeteus Pocock 1901 (Araneae: Mygalomorphae: Theraphosidae) from Brazil, with a new type of stridulatory organ. Zootaxa 1826, 45e58. https://doi.org/10.11646/ zootaxa.1826.1.3. ^nero Grammostola Bücherl, W., 1951. Estudos sobre a biologia e a sistem atica do ge Simon, 1892, vol. 1. Monografias, Instituto Butantan, pp. 1e203. rez-Miles, F., 2011. The species of Grammostola (AraFerretti, N., Pompozzi, G., Pe neae: Theraphosidae) from Central Argentina: taxonomy, distribution, and surface ultrastructure of coxal setae. Zootaxa 2828, 1e18. https://doi.org/10. 11646/zootaxa.2828.1.1. Gabriel, R., 2014. A new genus and species of theraphosid spider from El Salvador (Araneae: Theraphosidae). Br. Tarantula Soc. J. 29 (3), 146e153. Galleti-Lima, A., Guadanucci, J.P.L., 2018. Morphology of setae on coxae and trochanters of Theraphosinae spiders (Mygalomorphae; Theraphosidae). J. Arachnol. 46 (2), 214e225. https://doi.org/10.1636/JoA-S-17-021.1. n al conocimiento de TherGerschman De, P.B.S., Schialelli, R.D., 1966. Contribucio rias do aphosa leblondi (Latreille), 1804 (Aranea: Theraphosidae), vol. 33. Memo Instituto Butantan, pp. 667e674.
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