Kankuamo, a new theraphosid genus from Colombia (Araneae, Mygalomorphae), with a new type of urticating setae and divergent male genitalia

Abstract A new monotypic Theraphosidae genus, Kankuamo Perafán, Galvis & Pérez-Miles, gen. n., is described from Colombia, with a new type of urticating setae. These setae differ from others principally by having a small distal oval patch of lanceolate reversed barbs. Males of Kankuamo gen. n. additionally differ by having a palpal bulb organ very divergent from all known species, with many conspicuous keels dispersed across the median tegulum to the tip, mostly with serrated edges. Females differ by having spermathecae with a single notched receptacle, with two granulated lobes and several irregular sclerotized longitudinal striations. The new urticating setae, type VII, is characterized, illustrated and its releasing mechanism is discussed. It is hypothesized that these setae are the first in Theraphosinae subfamily whose release mechanism is by direct contact. Kankuamo gen. n. is described and illustrated on the basis of the type species Kankuamo marquezi Perafán, Galvis & Gutiérrez, sp. n., and their remarkable characteristics, morphological affinities and cladistic relationship are analyzed.


Introduction
Theraphosidae Thorell, 1869 is the most speciose of the Mygalomorphae with more than 130 genera and 980 species (World Spider Catalogue 2015), mainly distributed in the tropical and subtropical regions, and currently divided into 11 subfamilies (Guadanucci 2014). This family comprises large sized and setose spiders commonly known as tarantulas in the New World. A unique morphological characteristic of most New World theraphosids is the presence of defensive urticating setae (Cooke et al. 1972). This defense mechanism is found in roughly 540 of the 600 theraphosid Neotropical species (Bertani and Guadanucci 2013). Representatives of all known species of the subfamily Theraphosinae, as well as species of the Aviculariinae genera Avicularia Lamarck, 1818, Ephebopus Simon, 1892, Iridopelma Pocock, 1901, Pachistopelma Pocock, 1901, and Typhochlaena C.L. Koch, 1850 have urticating setae. The arboreal tarantulas Tapinauchenius Ausserer, 1871 and Psalmopoeus Pocock, 1895, and several 'Ischnocolinae' genera are the only New World theraphosids that lack any urticating setae (Bertani and Guadanucci 2013).
The morphological characteristics of urticating setae have long been used in taxonomy and systematics of Theraphosidae, being useful as a set of characters for differentiation of subfamilies and genera as shown in phylogenetic analysis (Cooke et al. 1972, Raven 1985, Pérez-Miles et al. 1996, Pérez-Miles 2002, Perafán 2010, Bertani and Guadanucci 2013. Six different types of urticating setae have been described based on their morphology, ornamentation, length and releasing mechanism; two types are known to Aviculariinae (II and V), and another four in Theraphosinae (I, III, IV and VI) (Cooke et al. 1972, Marshall and Uetz 1990, Pérez-Miles 1998) (see Bertani and Guadanucci 2013, Figure 1). Excepting type V which occurs on the distal prolateral surface of the palpal femora (Marshall andUetz 1990, Foelix et al. 2009), all other types are found on the dorsum of the abdomen.
During study of Colombian tarantulas, we discovered specimens from Sierra Nevada de Santa Marta, Colombia, with a different type of urticating setae which did not fit with any known types. These setae mainly differ by having a small distal patch of reversed lanceolate barbs (regarding the main barbs; sensu Cooke et al. 1972) near the penetrating tip, and small main barbs that extend along the whole seta from the apex. Additionally, males present a palpal bulb remarkably different from all known Theraphosidae, with a large number of conspicuous keels on tegulum and embolus. The morphology and arrangement of these keels do not easily fit with the palpal bulb homologies proposed by Bertani (2000) for the Theraphosinae. However, this taxon shares the main general characteristics of the Theraphosinae subfamily, extended subtegulum, keels on palpal bulb, and urticating setae (Raven 1985, Pérez-Miles et al. 1996. Females differ from other Theraphosinae genera by having spermathecae with a single notched receptacle with two granulated lobes and several irregular sclerotized longitudinal striations. Based on its unique combination of characters, we propose the new monotypic Theraphosinae genus Kankuamo Perafán, Galvis & Pérez-Miles, gen. n., which is here diagnosed, described and illustrated on the basis of the type species Kankuamo marquezi Perafán, Galvis and Gutiérrez sp. n. Morphological aspects are discussed and its phylogenetics relationship are analyzed based on a Theraphosinae cladistic re-analysis presented in this paper. Considering the size and morphology of the urticating setae in Kankuamo gen. n., we propose them as a novel type, here naming them as type VII urticating setae. These setae are described and illustrated, and their releasing mechanism is discussed.

Material and methods
Urticating setae terminology follows Cooke et al. (1972) and Bertani and Guadanucci (2013). Male palpal organ keel terminology follows Bertani (2000). Number and disposition of spines are enumerated from the anterior third to the posterior third, modified from Petrunkevitch (1925). Spination was recorded from the right-side limbs. All measurements were taken using an ocular micrometer and are given in millimeters (mm). Leg and palp measurements were taken on the dorsal side, along the central axis of the left-side limbs. The genital structures were removed for examination: either male left palpal bulb, or female spermathecae. Urticating setae from different regions of the abdomen were removed and examined under optical microscope and scanning electron microscope (SEM). Photographs were taken with an Infinity Lite camera adapted to the stereoscope lens (Nikon SMZ-10), SEM with JEOL JSM-5900 LV. The geographic coordinates are taken by GPS, Datum WGS84. The distribution map was produced using DIVA-GIS version 7.5.0 (http://www.diva-gis.org/). The type material is stored in 70% ethanol, and deposited in the Instituto de Ciencias Naturales Arachnological Collection (ICN-Ar), at the Universidad Nacional de Colombia, Bogotá, Colombia.
Abbreviations are listed below: Cladistic analysis. Cladistic analysis was based on the previous matrix of Theraphosinae genera used by Pérez-Miles et al. 1996 andPérez-Miles 2000 with some modifications. This matrix was completed as far as possible, including material examined and literature, complemented with the new evidences of the palpal bulb homology proposed by Bertani (2000Bertani ( , 2001, and the characters used on the matrix of Perafán and Pérez-Miles (2014). The original matrix from Pérez-Miles et al. 1996 was modified on characters referred to palpal bulb keels (characters 3 and 4) and replaced with those proposed by Bertani (2000Bertani ( , 2001, Perafán and Pérez-Miles (2014). Character 13 related to the spermathecae shape was also amended. Furthemore, the hypotehical outgroup used on the analysis previous (Pérez-Miles et al 1996, Pérez-Miles 2000 was modified by a Schismatothelinae taxon Guyruita Guadanucci et al., 2007(Guadanucci 2014) and the terminal Pseudotheraphosa Tinter, 1991 was eliminated because it's a junior synonym of Theraphosa Thorell, 1869 (Bertani 2001).

Results and discussion
Kankuamo Perafán, Galvis & Pérez-Miles, gen. n. http: Type species. Kankuamo marquezi Perafán, Galvis & Gutiérrez, sp. n. Diagnosis. Differs from all previously known genera of Theraphosidae by having a distinct type of urticating setae (Fig. 2, see description below), mainly characterized by having a small distal patch of lanceolated barbs arranged in reversed direction, regarding the main barbs, oriented with their tips towards the penetration tip (Fig. 2B). Male differs additionally from other genera by having a curved sub-conical palpal bulb with many conspicuous keels distributed throughout the majority of the subtegulum and embolus, especially developed on the dorsal and prolateral faces, most of them with serrated edges (Fig. 3E-3I). PSK, AcK, PIK, AK and SAK present (sensu Bertani 2000), additionally dorsal keels (DKs) and supra-accessory keels (SpAcK). Tibial apophysis on leg I absent (Fig. 3D). Females differ by having spermathecae with a single notched receptacle, with two granulated lobes, and several irregular sclerotized longitudinal striations (Fig. 3D).
Etymology. Kankuamo is a noun in apposition and refers to the indigenous people of the Chibcha family from the Caribbean region of Colombia, which inhabits the eastern slope of the Sierra Nevada de Santa Marta, whose language and culture are at endangered. Kankuamo gender is neuter.
Discussion. Morphology. Kankuamo gen. n. clearly presents the diagnostic characters of the subfamily Theraphosinae (Theraphosidae): male palpal bulb with large and extended subtegulum, and numerous developed keels; legs spinose, with normal scopulae on tarsi and presence of abdominal urticating setae (Figs 2-6). However, this  new genus differs dramatically from all previously known genera by their novel urticating setae type and the detailed morphology of male palpal bulb, making it difficult to establish their phylogenetic affinities. The general shape of the palpal bulb resembles approximately those of the genus Ami Pérez-Miles, 2008(mainly A. bladesi Pérez-Miles, Gabriel and Gallon, 2008and A. weinmanni Pérez-Miles, 2008, but with the subtegulum more elongated in Kankuamo gen n. (Fig. 3G). However, the transverse arrangement of bulb keels is also superficially similar to those of some Acanthoscurria Ausserer, 1871, or to the ring shaped keel of Hapalopus Ausserer, 1875. Numerous distal keels are also known in other genera such as Lasiodora C.L. Koch, 1850, Nhandu Lucas, 1983and Vitalius Lucas, Silva and Bertani, 1993, but the higher number of keels in Kankuamo gen. n. easily distinguish it from all those and other remaining genera in Theraphosinae. Furthermore, the high number of keels make difficult to establish homology with those of the family as Bertani (2000) suggested. Considering the extension and positional similarity we tentatively propose the presence of PSK, AcK, PIK, AK, and SAK, and we also propose the name supra-accessory keels (SpAcK) for those placed on proximal subtegulum (Fig. 3H, 3I) and dorsal keels (DKs) for those placed principally on dorsal face (Fig. 3E-3G). The absence of tibial apophysis is shared with Agnostopelma Pérez-Miles and Weinmann, 2010; Aguapanela Perafán, Cifuentes and Estrada, 2015; Metriopelma Becker, 1878; Nhandu; Sericopelma Ausserer, 1875 and Theraphosa Thorell, 1870.
Accordingly, Kankuamo gen. n. clearly differs from all genera of Theraphosidae known by the urticating setae type and male palpal bulb characters.
Cladistic relationship. A search using equal weights found 171 most parsimonious tress and the strict consensus of these did not provide any resolution. Search with implied weighting and different concavity indices (k = 3 to 12) found between 3 and 10 shortest trees. The strict consensus of each of these tress recovered different topologies, but between k = 8 and k = 12 the topology of strict consensus did not vary, for this reason we have selected this topology to test Kankuamo gen. n. affinities (Fig. 1). It is noted that this new phylogeny analysis of Theraphosinae is an incomplete analysis including only half of the Theraphosinae genera and relatively few characters.
Kankuamo gen. n. was resolved as the sister group of Metriopelma, supported by the character "femur IV without retrolateral scopula". Both genera also share the lack of the apophysis on males and a spermathecae with seminal receptacles completely fused. Kankuamo gen .n. presents a reversion to state (0) on character 16 "absence the urticating setae type I", a parallelism of the state (1) on character 31 "presence the accessory keels under prolateral inferior keel" and an autapomorphic character "presence of urticating setae type VII".
Distribution. See distribution of the genus (Fig. 7). Natural history. Kankuamo marquezi sp. n. inhabits Cuchilla San Lorenzo from Sierra Nevada de Santa Marta National Natural Park. Cuchilla San Lorenzo is located in its northwestern flank, in a gradient of altitude from 2000-2300 meters above sea level, in life zone of lower montane wet forest (Espinal and Montenegro 1963). The Sierra Nevada de Santa Marta is an isolated mountain range separated from the Andes chain. The locations where the specimens were collected are covered mainly by shrubby plants of the families Arecaceae and Chrysobalanaceae (Cuadrado-Peña 2005), and inhabited by the snakes genus Atractus and frogs of the genera Atelopus, and the endemic species from Sierra Nevada de Santa Marta Ikakogi tayrona and Geobatrachus walkeri (MG personal observations).  The similarities of the morphology and size of setae type VII with type II Aviculariinae (see Cooke et al. 1972, figs 11-12, 20-21) suggest a releasing mechanism by direct contact, as indicated for Avicularia (excepting A. versicolor, see Bertani et al. 2003), Iridopelma, Pachistopelma and Typhochlaena (Bertani 2012). These tarantulas direct the abdomen toward the stimulus and transfer the urticating setae when the abdomen of the spider touches against the target (Bertani and Marques 1996). Contrary, in Theraphosinae, the various types of urticating setae are dislodged by friction of the hind legs against the dorsum of the abdomen, and air-transported (Cooke et al. 1972). Bertani and Marques (1996) proposed that the differences of the shape, size and thickness between Theraphosinae and Aviculariinae urticating setae could explain the two releasing mechanisms. The morphological characteristics of all then known urticating hair types in Theraphosinae, particularly their light weight and aerodynamics, would allow them to float through the air. They suggest that short and/or thin setae with a ratio of 100:1 or 200:1 are able to float through the air. Bertani et al. (2003) compared length/width ratio between urticating setae type II and III and concluded that all airborne setae (including A. versicolor type II) are narrow, mean width ranging from 6 to 7 µm, and length/width ratio ranging from 98.3 to 208.3. In contrast, for urticating setae released by direct contact with the target, the width was greater, ranging from 15 to 22 µm and the length/width ratio of 37.3-46.8 (see Bertani et al. 2003, Table 1). Here, the newly described urticating setae type VII width is 33±3 µm and length/width ratio 34, which therefore represents the thickest setae known until now, and with the lowest known length/width ratio, aspects that together reinforce the hypothesis that their releasing mechanism is by direct contact.
Furthermore, the larger size, broader shape and often dispersed arrangement of many of the main barbs of Theraphosinae urticating setae (types I, III, VI, VI) involved in their urticating effects, presumably often contribute their ability to float in the air. Conversely, in both urticating setae types II and VII, which presumably do not float by air, the main barbs are only residual denticles, being much smaller than those of other types with known air dispersal.
The differences in the position of the penetrating tip between type VII and II also suggest a different penetration mechanism. On first contact with the target, the urticating setae type II pivots on its stalk so the apical end moves away from the target, while the basal penetration tip (which is actually adjacent to the stalk) instead lodges into the target as the stalk releases (see Bertani and Marques 1996, fig. 3). By contrast, the apical penetrating tip of the urticating setae type VII (at the opposite end to the stalk) is able to penetrate directly into the target, at the first contact with the object.
Experimentally, the possible mechanism of action of the urticating setae type VII was observed while handling specimens in alcohol. These urticating setae easily perforated the skin of human fingers perpendicularly. The dorsum of the tarantula's abdomen was touched intentionally, and on further examination of the affected fingers with a stereoscope microscope, many of these setae were found embedded in the skin (Fig. 8). These setae were firmly fixed into the skin but none penetrated more than a third of their length. When we attempted to remove them from the skin, these setae were easily broken at their distal end. Based on our observations together, our assumption is that the patch of the lanceolate reverse barbs in the distal area can serve as a breakpoint into the skin, ensuring that the apex of the seta remains within the tissues.
Another aspect to consider is that all previously known species with contact urticating setae have leg spines absent or reduced (Cooke et al. 1972, Bertani et al. 2003. Kankuamo gen. n. therefore represents a remarkable exception to this suggested character association because, in contrast, they do have legs with some spines, mainly conspicuous on metatarsi and tarsi of hind-leg pair IV. Leg spines in other theraphosids have been functionally associated with releasing of airborne urticating setae by facilitating rubbing, with the exception of A. versicolor which use claw tufts for rubbing (Cooke et al. 1972, Bertani and Marques 1996, Bertani and Guadanucci 2013. One possible interpretation is the plesiomorphic retention of contact setae could reflect a close phylogenetic relationship between Aviculariinae and Theraphosinae, as proposed by Pérez-Miles et al. (1996). In this scenario, the additional development of posterior leg spines (such as found in Kankuamo gen. n.) could have facilitated the evolutionary transformation of contact setae into derived airborne setae, which are now widespread among Theraphosinae. However, if contact setae are interpreted as derived, the posterior leg spines present in Kankuamo gen n. could be regarded as a plesiomorphic retention.

Conclusions
Kankuamo gen. n. fits the diagnostic characters of Theraphosinae, but also shows a very divergent palpal bulb morphology and the presence of a new abdominal urticating setae type. These setae are unique, and here are proposed to be the only contact released urticating setae yet known within the Theraphosinae, although this release mechanism was previously well known only for Aviculariinae. Also, the supernumerary keels on the male palpal bulb clearly distinguish it from all known theraphosid species. Kankuamo gen. n. was resolved as the sister group of Metriopelma on our preferred phylogeny of Theraphosinae.