Two new species of Bonnetina tarantulas (Theraphosidae: Theraphosinae) from Mexico: contributions to morphological nomenclature and molecular characterization of types

Two new species of tarantulas from Mexico are described and illustrated: Bonnetina tenuiverpis and Bonnetina juxtantricola, from the states of Mexico and Guerrero, respectively. Male palpal bulbs, tibial apophyses and spermatheca are among the most taxonomically informative characters. Male bulb microstructure is revealed from scanning electron microscopy; both new and homologous structures to other Theraphosinae genera are identified and described. Nomenclatural changes for male tibial apophyses are also proposed. The holotype male and allotype female of one of the species are molecularly characterized and matched from CO1 partial sequence. http://zoobank.org/urn:lsid:zoobank.org:pub:BBDBBEDE-6983-4F5C-B34C-4622DF494C84


Introduction
Bonnetina Vol 2000 is one of the most recently described genera of Mexican tarantulas and is also endemic to the country. Six species have been described since its original report: Here we present the description and wide illustration of two new species of Bonnetina, the initial results of author DO's PhD project on phylogenetic systematics of the genus.
We also propose corrections to the nomenclature of the males leg I tibial spurs introduced in preceding papers on Bonnetina and we make a deep examination of male palpal bulbs (from scanning electron microscope [SEM] images), that reveal structures apparently homologous to those of other Theraphosinae genera, but also others not previously recorded and that are thus described and named here for the first time.
To expand the descriptions from the traditional morphological only approach to potentially useful molecular characterization, we include a cytochrome c oxidase subunit 1 (CO1) ≈ 960 bp partial sequence of the holotype and allotype of one of the new species Spermathecae were cleaned by submerging them in absolute lactic acid from one to several hours.
The term allotype is here considered for the fully described paratype female, which assures an easy identification of the specimen, although it is a term not regulated by the current International Code of Zoological Nomenclature (1999).
Pedipalpal bulbs terminology follow Bertani (2000) in general terms. Nevertheless, there are some keels found in the species treated in this paper that do not seem to be homologous to any of those described before and are thus named here for the first time (Figures 4,11). Sperm pore keels are two structures located at the ventral apex of the embolus that fold onto each other, shaping the sperm outlet as a sperm pore. The dorsal keel is a structure located subapically, opposite to the sperm pore, in one of the species treated in this paper. The prolateral sub-apical keel, in the other species, is similar in shape and position to the dorsal keel, but is located between prolateral superior keel and sperm pore, so should not be considered a priori homologous to the dorsal keel. Future thorough exploration of several species of Bonnetina and related genera is expected to help clarify the homology of bulbal structures. The position of the structures is considered discarding the torsions that the embolus presents from base to apex, using its base as a reference.
Terminology for urticating hairs was taken from Cooke et al. (1972); for tarsal scopulae from Pérez-Miles (1994); for spination patterns from Petrunkevitch (1925) and for barbed hairs from Font Quer (1953) (i.e. if barbs are at least two times longer than hair width, it is plumose; if shorter, it is ciliate).
Material examined will be deposited in the following collections: CNAN and American Museum of Natural History, New York, USA (AMNH).

Molecular protocol
Immediately after the specimens were sacrificed, right leg III was removed and preserved in 96% ethanol at -20°C. DNA extraction was performed with Qiagen DNeasy Tissue Kit (Qiagen, Venlo, Limburg, Netherlands) and its products were stored at -20°C until amplification.
Sequencing was performed at Instituto de Biología (UNAM) in a 3500 XL Genetic Analyzer (Life Technologies), using the same manufacturer's BigDye® Terminator v3.1 cycle sequencing kit.
The reading frame was determined by translating to the six possibilities using the Expasy Online Translate Tool (http://web.expasy.org/translate/). The only option that did not have stop codons was then successfully aligned with other theraphosid spiders using GenBank's Standard Protein Blast, which confirmed the selection. Sequences are also deposited at GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Allotype. ♀ (CNAN-T0756): same collecting data as holotype.

Etymology
The specific name is composed by the Latin adjective tenuis (slender) and plural noun verpis (penes). It makes reference to the attenuate condition of the male pedipalpal bulbs of this species.

Diagnosis
Males differ from those of all known species of the genus by the shape of its palpal bulbs, that are also remarkably slender compared to those of the other species. It additionally has much better developed tibiae I accessory apophyses than B. alagoni and B. aviae. It is a distinctly smaller species than B. cyaneifemur, B. rudloffi, B. papalutlensis and B. tanzeri. Females differ from those of other species by having subdigitiform spermatheca instead of domiform (as in B. alagoni and B. aviae), flattened (B. tanzeri), subtriangular (B. papalutlensis) or digitiform (B. cyaneifemur). Females of B. rudloffi have not been described.

Description
Male holotype Morphology Some quantitative characters are given in Table 1  Coloration and pilosity. Carapace covered by dense shiny coppery pubescence, that masks partially the dark brown colour of the integument ( Figures 1A, 2A). Posterior area of carapace bears distinctly thick erect setae ( Figure 2B). Leg and pedipalpal segments (except femora) with intermixed copper and light brown setae. Femora black, with iridescent blue tones. Abdomen dark brown, with long and thick red setae and dense short dark brown setae. Carapace with caput nearly flat and fovea broad and procurved. Ocular area: eight eyes disposed in two rows on markedly elevated tubercle (PLE almost perpendicular to carapace) ( Figure 2C); anterior eye row procurved; posterior row, recurved ( Figure 2D). Ocular quadrangle width, 1.14; length, 0.86. Clypeus, 0.14 wide. AME circular, diameter, 0.28; ALE ovoid, greater diameter, 0.36; PME ovoid, greater  Chelicerae with seven teeth (left appendage), and seven teeth plus one tiny basalmost tooth (right), close and parallel to the promargin on ventral side. Table 1. Bonnetina tenuiverpis sp. nov. and Bonnetina juxtantricola sp. nov. Variation of some quantitative characters in the specimens of the type series. Measurements in mm. PLS: posterior-lateral spinnerets. UHP/A: urticating hairs patch length/abdomen length. PA(AA)/RA: length of the prolateral(accessory)/retrolateral tibiae I apophyses. Except in column 4, hyphens represent the separation between the value of each character in the left appendage (at left) and the right one. In column 4, commas separate the values in AMNH and CNAN-T0757 paratypes. Sternum ( Figure 2E) slightly convex to its centre, covered uniformly by erect thick hairs and other hairs much smaller; with three pairs of sigillae, placed opposite to coxae I, II and III. Labium subtrapezoidal ( Figure 2F); middle length, 0.90; anterior width, 0.80; posterior width, 1.25.
Retrolateral face of palpal tibiae with prominent, apically inclined conicshaped nodule near apex, densely covered by long and thick setae ( Figure 2G).
Palpal bulbs (Figures 3, 4) with embolus very slender, with its apical half markedly curved and twisted dorsally and retrolaterally (from base to apex) to the point that in the apex, the ventral structures of the bulb become prolateral. Prolateral inferior, prolateral superior, sperm pore and dorsal keels present. PI keel is mostly serrated and extends from the base of the embolus to half of the space between its most apical denticle and the sperm pore. PS keel extends from close to the base of the embolus to sperm pore level. SP keels extend from the bulb apex to the sperm pore and are folded onto each other, except in the basalmost region, forming the sperm pore; they are not completely fused at the apex, allowing a small pore at embolus tip. D keel, about as long as SP keels, is subapical to the embolus.
Femora of palps and legs I and II prolaterally and femora of legs IV retrolaterally covered by pad of simple and ciliated hairs.
Scopulae Metatarsi. On legs I entire segment except apex; on legs II apical 5/6; on legs III distal half of segment and on legs IV apical 1/5. Tarsi. On legs I and II entire with few dispersed type B hairs; on legs III divided completely by a band of 1-3 fine hairs; on tarsi IV divided full length by band of 3-4 very thick hairs. Very dense claw tufts on every leg.
Abdominal urticating hairs. Type III, in dorsal oval patch, located mostly in posterior half of abdomen, covering 0.42 of its length.

Molecular characterization
Mitochondrial CO1 partial sequence (GenBank accession: KC807369): g ttg cct cct tcg ttg ttt tta tta gta tta tct tct ttg act gat gta ggg gtt ggg gct ggg tga act att tat ccg cct tta tct tct ttt atg ggt cat tct ggt gga ggg atg gat ttt gct att ttt tct ttg cat ttg gct ggg gct tcg tct att atg ggg tct gta aat ttt att act act gtt ata aat atg cgt gca tct gga ata tcg atg gaa cgt att cct ttg ttt gtt tga tct gta gtg att aca act gta tta ttg tta tta tcg tta ccg gtg ttg gct ggg gct att act ata tta ttg tcg gat cgg aat ttt aat act tct ttt ttt gac ccg gct ggt ggt ggg gat cct att ttg ttt cag cat tta ttt tga ttt ttt gga cat ccg gag gta tat att ttg att tta cca gga ttt gga atg gta tct cat att att agt tct tct gtt ggt aaa cgt gaa cct ttt gga tct tta gga ata att tat gct atg gtt aga att ggt gga ata gga ttt gtt gtg tga gca cat cat atg ttt tct gtt ggt ata gat gtt gat act cgt gca tat ttt act gcg gca act ata gtg att gct gta cct acg gga att aag gtt ttt aga tga ata gca acg ttg tat gga tca tat ttt aag ttg gat gtg tct ttg atg tgg tgt att gga ttt gta ttt tta ttt act atg ggt gga ttg act ggg gta gta tta gct aat tct tct ttg gat att gtg ttg cat gat acg tat tat gtt gtt gct cat ttt cat tat gtg ttg aga atg gga gct gta ttt gct att atg ggg ggt ttg gct tat tga ttt cct tta ttt ttt gga act ata ata aat ccn nnn tta atg aag ttg cag ttt gtt ata atg ttt gtt ggt gta aat tta act

Preservation state
The specimen is in optimal conditions, stored in a flask of 80% ethanol. Right leg III preserved in 96% ethanol at -20°C for molecular studies. Left pedipalpal bulb is housed apart, coated with gold.

Female allotype
Morphology Some quantitative characters are given in Table 1 Coloration and pilosity. As for holotype, but the carapace pubescence is less dense and the femora lack blue tones ( Figures 1B, 6A). Posterior area of carapace bears distinct setae, considerably thinner than those on the holotype ( Figure 6B).
Chelicerae with nine teeth plus one tiny basalmost tooth (left appendage) and eight teeth plus one tiny basalmost tooth (right), close and parallel to the promargin on ventral side. Sternum ( Figure 6D) slightly convex, covered uniformly by thick erect hairs and other hairs much smaller; with three pairs of sigillae, placed opposite to coxae I, II and III. Labium subtrapezoidal; middle length, 1.25; anterior width, 1.15; posterior width, 2.05 ( Figure 6E).
Scopulae. Metatarsi. On legs I entire; on legs II entire except base; on legs III apical half prolaterally and apical third retrolaterally ( Figure 6F); on legs IV apical fourth. Tarsal scopulae and femoral pads as on holotype.
Coxae and trochanters of palps and legs I and II as in holotype, covered by nonplumose hairs.
Abdominal urticating hairs. Type III, in oval dorsal patch (with rear part distinctly narrower), located mostly in posterior half of abdomen, covering 0.43 of its length.
Only one spermatheca subdigitiform ( Figure 7A, B), symmetrical (in dorsal and ventral views) with well-defined base, neck and fundus; length, 1.02; base width, 0.72. A wide poorly sclerotized atrium is at its base.

Molecular characterization
Mitochondrial CO1 partial sequence (GenBank accession: KC807370): ttg ttg ccn cct tcg ttg ttt tta tta nta tta tct tct ttn act gat gta ggg gtt ggg gct ggg tga act att tat ccg cct tta tct tct ttt atg ggt cat tct ggt gga ggg atg gat ttt gct att ttt tct ttg cat ttg gct ggg gct tcg tct att atg ggg tct gta aat ttt att act act gtt ata aat atg cgt gca tct gga ata tcg atg gaa cgt att cct ttg ttt gtt tga tct gta gtg att aca act gta tta ttg tta tta tcg tta ccg gtg ttg gct ggg gct att act ata tta ttg tcg gat cgg aat ttt aat act tct ttt ttt gac ccg gct ggt ggt ggg gat cct att ttg ttt cag cat tta ttt tga ttt ttt gga cat ccg gag gta tat att ttg att tta cca gga ttt gga atg gta tct cat att att agt tct tct gtt ggt aaa cgt gaa cct ttt gga tct tta gga ata att tat gct atg gtt aga att ggt gga ata gga ttt gtt gtg tga gca cat cat atg ttt tct gtt ggt ata gat gtt gat act cgt gca tat ttt act gcg gca act ata gtg att gct gta cct acg gga att aag gtt ttt aga tga ata gca acg ttg tat gga tca tat ttt aag ttg gat gtg tct ttg atg tgg tgt att gga ttt gta ttt tta ttt act atg ggt gga ttg act ggg gta gta tta gct aat tct tct ttg gat att gtg ttg cat gat acg tat tat gtt gtt gct cat ttt cat tat gtg ttg aga atg gga gct gta ttt gct att atg ggg ggt ttg gct tat tga ttt cct tta ttt ttt gga act ata ata aat ccn nnn tta atg aag ttn cng ttt gtt ata atn ttt gtt ggn gta aat ata act

Preservation state
The specimen is in optimal conditions, stored in a flask with 80% ethanol. Right leg III preserved in 96% ethanol at -20°C for molecular studies. Genital area is housed in a plastic vial inside the flask.

Variation
The variation of some quantitative characters in the three females examined is summarized in Tables 1 and 2. None of the females has thick erect setae on the posterior margin of carapace, although setation in this area is distinct from the rest of carapace.
All have a row of erect hairs that extends between ocular area and fovea. The shape of the urticating hairs patch varies, although it is oval for all specimens.
Coxae and trochanters lack plumose setae and hairs in all specimens. The pubescence of the carapace is considerably denser on the male than on the three females. The coloration is very similar in the two females that were examined alive.
Spermathecae of the three specimens share the same structural pattern (Figure 7), although that of AMNH paratype is wider. The AMNH paratype also has tarsi on all the legs divided, probably due to its smaller size (Pérez-Miles 1994).
The holotype and allotype differ only by one base pair of the CO1 sequenced region (0.1% uncorrected pairwise sequence divergence).

Distribution
Bonnetina tenuiverpis is known only from the vicinity of Zuluapan town, situated in the State of Mexico and geographically at 1250 m asl in the Trans-Mexican Volcanic Belt (Eje Neovolcánico), which extends from Pacific to Atlantic coast of Central-Southern Mexico.

Natural history
The specimens looked faded when collected in August 2012, and all moulted in captivity in September and October. The only male matured on 11 October, suggesting that the breeding season might include fall. The only known population lives in an absolutely open, very rocky grassland (Figure 8) that is frequently used by cattle. Dominant vegetation of the area is tropical deciduous forest. The specimens were found mainly on the edges of middle sized rocks (20-150 cm diameter), in shallow depressions bordered by silk, presumably dug by them. Two specimens of the tarantula Brachypelma cf. albiceps, three of another Bonnetina species and few of Diplocentrus scorpions were also found syntopically. These three species could be competing for resources or even preying on B. tenuiverpis.

Etymology
The specific name is composed from the Latin preposition juxta (nearby), the noun antrum (cavern) and the suffix cola (dweller). The name can be translated as 'nearbycavern dweller' and makes reference to the type locality of this species, which is the surroundings of an archaeologically very valuable cave.

Diagnosis
Males differ from those of all known Bonnetina species in the shape of the palpal bulbs. Additionally, from B. rudloffi, B. alagoni, B. papalutlensis, B. tenuiverpis and  B. tanzeri in that PI bulbal keel is not denticulate as on those species. It also differs from males of B. aviae in the lesser retrolateral bend of the embolus, which describes an angle of approximately 30°with the axis of the bulb and about 70°in B. aviae. The male tibia I accessory apophysis is present but considerably less developed than that of B. cyaneifemur, B. rudloffi, B. papalutlensis, B. tanzeri and B. tenuiverpis. Females have domiform spermatheca and so differ from the species that have it digitiform (B. cyaneifemur), subdigitiform (B. tenuiverpis), subtriangular (B. papalutlensis) or flattened (B. tanzeri). Females differ from those of B. aviae and B. alagoni by presenting very distinctly thick, erect setae on the posterior area of carapace.
Chelicerae with seven teeth (left appendage) and seven teeth plus one tiny apicalmost tooth (right), close and parallel to the promargin of ventral side.
Sternum ( Figure 9E) slightly convex to its centre, covered uniformly by thick erect hairs and other hairs much smaller; with three pairs of sigillae, placed opposite to coxae I, II and III. Labium subtrapezoidal; middle length, 0.96; anterior width, 0.90; posterior width, 1.70 ( Figure 9F).
Palpal bulbs (Figures 10, 11) robust with thick embolus, that is moderately curved and twisted dorsally and retrolaterally (from base to apex) so that in the apex, the ventral structures of the bulb become prolateral. Prolateral inferior, prolateral superior, sperm pore and prolateral sub-apical keels present. PI keel is smooth and extends from close to the base of the embolus to near to sperm pore; it ends sharply at its apex, forming a distinct prominence. PS keel extends from embolus base to apex. SP keels extend from the bulb apex to the sperm pore and are folded onto each other, except in the basalmost region; they are fused at the apex, not forming a pore at the embolus tip. PSA keel, about as long as SP keels, is subapical to the embolus and located between PS and sperm pore.
Legs I holding organ. Tibiae I with three apophyses near the apex, all parallel to segment axis ( Figure 12A, B). Prolateral apophysis tubular, blunt at its apex and bearing a megaspine on its internal border; length, 0.48/0.48 (left/right). Retrolateral apophysis curved, acute at apex; length, 1.20/1.10. Accessory apophysis very poorly developed, bearing a subtriangular megaspine at its apex and a spine on the internal border; length, 0.14/0.10. When flexed, the moderately curved metatarsus I ( Figure  12C) folds between prolateral and retrolateral apophyses. Metatarsi I with nodule of 16 (left) and 19 (right) granules on basal ventro-retrolateral region ( Figure 12B). Two (left) and one (right) small rounded structures, presumably modified spines, on the basal ventro-prolateral region of metatarsi I ( Figure 12A, C).
Femora of palps and legs I and II prolaterally and femora of legs IV retrolaterally covered by pads of ciliated hairs.
Scopulae Metatarsi. On legs I entire segment except the basal 1/5; on legs II apical 2/3; on legs III on apical half (left) and apical 1/3 (right) of segment; on legs IV apical 1/5. Tarsi. On legs I and II entire with few dispersed type B hairs; on legs III divided completely by a band of 1-3 fine hairs; on legs IV divided full length by band of 2-4 very thick hairs. Claw tufts dense on every leg.
Abdominal urticating hairs. Type III, in patch located dorsally in central-posterior half of abdomen, covering 0.31 of its length. It could have been oval or subrectangular, according to the mark, but only few hairs remain, located on its anterior border.

Preservation state
The specimen is in good condition, stored in a flask of 80% ethanol. Both pedipalpal bulbs are housed apart, coated with gold. The specimen is homogeneously reddishbrown due to the length of time it has remain preserved.
Abdominal urticating hairs. Type III, in oval dorsal patch (with rear part moderately narrower), located dorsally on central-posterior half of abdomen, covering 0.33 of its length.

Preservation state
The specimen is in good condition, stored in a flask of 80% ethanol. Genital area is housed in a plastic vial inside the flask. The specimen is homogeneously reddishbrown, due to the length of time it has remain preserved.

Distribution
Bonnetina juxtantricola is known only from the vicinity of Juxtlahuaca cavern, situated in Guerrero state and geographically at 930 m asl in the Sierra Madre del Sur, a mountain range that extends parallel to the Pacific Ocean coast between Central and Southern Mexico.

Natural history
Very little is known of the natural history of this species. The only known male was collected in September, suggesting that this month could be part of its breeding season. Both male and female were collected under stones in a tropical deciduous forest.

Discussion
One of the main challenges that this study faced was determining the homology and therefore the appropriate nomenclature for the male pedipalpal bulbs' structures of the new species described. These organs are fundamental in theraphosine spiders' taxonomy as they are very often used as the cornerstone to differentiate between genera and species in the group. By using SEM images, it was possible to find a considerable amount of information in the two new Bonnetina species. Nevertheless, the genus has never been included in phylogenetic or comparative morphology analyses, and belongs to a subfamily of more than 50 genera (Ortiz 2008) with unclear relationships. That fact, joined to the wide diversity of bulbal structures in the subfamily (Bertani 2000), makes it very hard to apply the congruence test, which is the most decisive homology test as it helps to discriminate homology from homoplasy (Patterson 1982).
In the description of Bonnetina papalutlensis, Mendoza-Marroquín (2012) mentioned the presence of a Subapical denticulate keel (sensu Bertani 2000). That keel seems to be clearly homologous to the keel that we name here as prolateral inferior, which is also denticulate in B. tenuiverpis. Bertani (2000) found that prolateral superior and prolateral inferior keels are present in most of the 27 studied genera of Theraphosinae and he defined them as 'two parallel keels … in the prolateral area of the embolus'. This is exactly how the two longest keels on the bulbs of B. tenuiverpis and B. juxtantricola look. Additionally, subapical keel was found always associated to apical keel, which is probably absent in B. juxtantricola and definitely absent in B. tenuiverpis. After Bertani (2000), other authors have considered prolateral superior and prolateral inferior as the basic keels in Theraphosinae (e.g. Fukushima et al. 2008;Pérez-Miles et al. 2008;Yamamoto et al. 2012).
Concerning the keels that are described for the first time in this contribution, there is no evidence to homologize dorsal and prolateral sub-apical keels to any of the already recognized and named keels. Although prolateral sub-apical keel could be alleged to be positionally similar to apical keel, and dorsal keel to retrolateral keel, in the context of phylogenetic uncertainty, there are important objections to assume their homology: (1) apical keel is not as common in Theraphosinae as prolateral superior and prolateral inferior keels, while retrolateral keel is only present in a small number of genera (Bertani 2000); (2) the low structural complexity of these keels is likely to hide independent acquisitions (homoplasy); and (3) each of these keels is present in only one of the species of Bonnetina herein described, revealing variation and thus remarkable homoplasy even at generic level.
Assigning names to structures based on very weak evidence is equivalent to assuming homology arbitrarily and, for example, can lead to erroneous character scoring and conclusions in morphological phylogenetic analyses. Not giving names to these structures is a serious impediment to their use in the taxonomy of the group; this is not desirable, considering the noticeable differences that are found among both species herein described and other Bonnetina species (Ortiz & Francke, unpublished observations). We conclude that the more reasonable choice is to consider them autapomorphic and thus give new names to keels that are especially difficult to homologize to those already described. If in the future Theraphosinae bulbal structures are clarified and it is found that prolateral sub-apical and dorsal keels are homologous to other structures previously named, it will be easy to modify Bonnetina bulbal nomenclature in consequence.
On the other hand, we think that there is no doubt of the authenticity of sperm pore keels, as nothing similar to those structures has been described before.