Ultrastructure of the Sexually Dimorphic Basitarsal Glands of Leg I in Manaosbiid Harvestmen (Opiliones, Laniatores)

JOURNAL OF MORPHOLOGY 272:872–882 (2011)

Daniel N. Proud* and Bruce E. Felgenhauer

Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana

ABSTRACT Chemical communication is an important mate choice, intrasexual contests, locating mates, aspect of arthropod biology especially for those arthro- defense from predators, and species recognition; pods with limited abilities to detect visual and acoustic Andersson, 1994). Sexually dimorphic glands are signals. Sexually dimorphic glands are often associated associated with the secretion of chemical substan- with the production of pheromones, which play a role in ces such as pheromones that may function in reproductive processes. In the family Manaosbiidae (Opi- liones: Laniatores), males exhibit an enlarged, swollen, attracting and/or stimulating mates, marking ter- often fused, and spindle-like basitarsus on leg I. In this ritories or trails, or recognizing or alarming con- study, we provide a novel description of the morphology specifics (Wyatt, 2003). and ultrastructure of the glandular structures found in Chemical signaling plays a vital role in both the proximal swollen tarsomeres of the male manaosbiid interspecific and intraspecific communication Rhopalocranaus albilineatus Roewer, 1932 and compare across many taxa (Wyatt, 2003) but is particularly the external leg I morphology with that of two other important for arthropods, which have limited use manaosbiid harvestmen (Barrona williamsi Goodnight of visual and acoustic signals due to constraints and Goodnight, 1942 and Cranellus montgomeryi Good- imposed by their small body size (Greenfield, 2002). night and Goodnight, 1947). The two proximal tarso- In arachnids, chemical signals associated with meres of the male R. albilineatus leg I contain two large, paired, acinar glands consisting of many glandular cells. reproductive behaviors have been observed in the Cells empty their secretory products into a large, orders Acari (Kuwahara, 2004; Sonenshine, 2006), branched epicuticular duct, which exits the leg via a Araneae (see review in Gaskett, 2007), Opiliones pore on the ventral region. Thus, a total of four glandu- (Martens, 1969), and Scorpiones (Polis and Sissom, lar structures are present within the two swollen tarso- 1990; Gaffin and Brownell, 1992, 2000). Best stud- meres and each possesses a conducting canal and pore. ied in spiders sex pheromones are often produced Finally, we discuss possible roles of these basitarsal by females and may be emitted from the web silk or glands in manaosbiid reproductive biology based on the through cuticular glands (Gaskett, 2007). Despite present understanding of sexually dimorphic glands our understanding of the chemical composition and in other terrestrial arthropods (i.e., insects and spiders). behavioral effects of spider pheromones, the glands J. Morphol. 272:872–882, 2011. Ó 2011 Wiley-Liss, Inc. that produce these hormones remain poorly under- KEY WORDS: sexual dimorphism; glands; histology; stood. In Opiliones, the use of sex pheromones is reproductive biology; arachnid largely unknown and evidence for chemical communication in sexual encounters is limited (Sˇ ilhavy´, 1967; Martens, 1969; Schulz, 2004; Shultz, 2005). The best studied glandular structures in harvestmen are the repugnatorial glands, which are INTRODUCTION also called scent glands, odoriferous glands, or stink glands, and are a key synapomorphy of Opi- Secondary sexually dimorphic traits, observable liones (Gnaspini and Hara, 2007). Several studies as differences in body size, coloration, or anatomi- have examined the morphology and ultrastructure cal structures, are often shaped by sexual selec- of the glands (Gutjahr et al., 2005; Schaider and tion (Andersson, 1994). Such sexually dimorphic traits are exhibited by many diverse arthropod *Correspondence to: Daniel N. Proud; Department of Biology, groups, e.g., beetles (Kawano, 2006; Pomfret and University of Louisiana at Lafayette, 300 E St Mary Blvd, Lafay- Knell, 2006), fiddler crabs (Oliveira and Custodio, ette, LA 70504-2451. E-mail: [email protected] 1998; Murai and Blackwell, 2006), myriapods (Conde´ and Nguyen Duy-Jacquemin, 1992; Pe- Received 22 September 2010; Revised 1 December 2010; reira, 1999), and spiders (Framenau and Hebets, Accepted 13 February 2011 2007; Aisenberg et al., 2010). The functional sig- Published online 28 April 2011 in nificance of sexually dimorphic traits has been Wiley Online Library (wileyonlinelibrary.com) explained by several general mechanisms (e.g., DOI: 10.1002/jmor.10954

Ó 2011 WILEY-LISS, INC. SEXUALLY DIMORPHIC BASITARSAL GLANDS IN MANAOSBIIDS 873 Raspotnig, 2009). Others have mapped morphologi- 2011). In males of the manaosbiid Isocranaus stri- cal and behavioral characters associated with the natii Sˇ ilhavy´, 1979 the first two basitarsal seg- repugnatorial glands on to hypothesized phyloge- ments are enlarged and fused and have three netic trees to demonstrate the potential use of large glandular openings approximately 15 lmin these defensive characters in resolving phyloge- diameter along the ventral region (Willemart netic relationships (Hara and Gnaspini, 2003). et al., 2010). Despite the general understanding of the repugna- In this study, we examined the morphology and torial glands, there is little information regarding ultrastructure of the sexually dimorphic basitarsus smaller tegumental glands (but see Gutjahr et al., on leg I of the manaosbiid harvestman Rhopaloc- 2005) and sexually dimorphic glands for which the ranaus albilineatus Roewer, 1932 using light microanatomy, ultrastructure, and functional sig- microscopy (LM), scanning (SEM), and transmis- nificance are poorly understood. sion electron microscopy (TEM). Additionally, Cra- Many species of harvestmen (Arachnida, Opi- nellus montgomeryi Goodnight and Goodnight, liones) posses sexually dimorphic structures includ- 1947 and Barrona williamsi Goodnight and Good- ing enlarged chelicerae, increased armature on leg night, 1942 were examined using SEM to compare IV, increased leg length, enlarged leg segments, external morphology of the glandular openings glandular structures, and rows of spines (Pinto-da- and sensory structures on the basitarsus of leg I. Rocha and Giribet, 2007). In cyphophthalmid har- Aside from species descriptions for manaosbiid vestmen, some examples include the tarsal glands harvestmen, which describe leg I basitarsus as on leg IV associated with the adenostyle found in swollen, enlarged, and spindle-like, only one study most families (Martens, 1979, Shear, 1993) and the has examined the sexually dimorphic leg in anal or sternal pore glands found in males of enough detail to find glandular openings on the all cyphophthalmid families (Pinto-da-Rocha and tarsomeres of one species (Willemart et al., 2010). Giribet, 2007). Other sexually dimorphic structures We provide a detailed description of the morphol- are described for cyphophthalmid harvestmen but ogy and ultrastructure of the glands found within are generally known only for individual genera or the basitarsus of leg I in males of R. albilineatus, species (see review in Willemart and Giribet, 2010). a comparison of the external morphology of leg I In several Dyspnoi taxa, males possess sexually in three manaosbiids and discuss possible roles of dimorphic cheliceral glands from which females the glands in the behavioral ecology and reproduc- obtain a secretion before or during copulation tive biology of these harvestmen. (Martens, 1969, 1973; Martens and Schawaller, 1977). In the suborder Eupnoi, the cheliceral horns and longer pedipalps of males are thought to assist MATERIALS AND METHODS in both intrasexual (i.e., male–male contests) and intersexual (i.e., grasping the female) interactions During summer 2008, we collected two species of manaosbiid harvestmen from Trinidad (R. albilineatus and C. montgomeryi) (Willemart et al., 2006). Many sexually dimorphic and examined the enlarged basitarsus of leg I using LM, SEM, structures have been described for the highly and TEM. A third species, B. williamsi, was collected from diverse suborder of Laniatores (Pinto-da-Rocha and Panama in September 2009 and examined. Length and diame- Giribet, 2007). Increased armature on leg IV in ter of the two proximal segments of the basitarsus of males and females of R. albilineatus (10 males; 10 females), C. montgo- some gonyleptids has been shown to function as a meryi (15 males; 10 females), and B. williamsi (three males; weapon in intraspecific male–male contests (Willemart three females) were measured using a digital micrometer. Data et al., 2009). In many cosmetid harvestmen, males were normally distributed; therefore, we employed Welch’s exhibit an enlarged basitarsus on leg I, enlarged t test to compare means. SEM was used to examine external chelicerae, and/or increased armature on leg IV morphology of the glandular openings and sensory structures on the basitarsus of leg I. For orientation purposes, LM was (Townsend et al., 2010). In a scanning electron mi- used to determine the presence and location of the gland in leg croscopy (SEM) survey, sexually dimorphic tegu- I through serial sections in both cross section and sagittal mental gland openings found on legs I, III, and IV section, and TEM was used to examine ultrastructure of the were described for 16 species of laniatorean glands. For SEM, specimens were fixed in 70% ethanol and dehy- harvestmen representing eight families (Willemart drated in a graded ethanol series to 100%. Specimens were et al., 2010); however, histology of the glands was chemically dried using hexamethydisilzane, sputter-coated with not examined. gold (20 nm), and examined at an accelerating voltage of 15 The Manaosbiidae is a relatively small family of kV using a Hitachi S-3000N scanning electron microscope. For TEM, specimens were fixed in Trump’s fixative (a mixture harvestmen that is distributed throughout the 2 of formalin and glutaraldehyde in 0.2 mol l 1 sodium cacodylate northern parts of South America into Central buffer, pH 7.4). Legs were dissected, rinsed in 0.2 mol l21 so- America and throughout parts of the Lesser Antil- dium cacodylate, postfixed in 2% OsO4 for 90 min at room tem- les (Kury, 2003, 2007; Townsend et al., 2011). perature, dehydrated in a graded ethanol series to 100% and Within Manaosbiidae, males of most species transferred to 100% acetone. Specimens were then infiltrated and embedded in Spurr’s low viscosity resin (Spurr, 1969) and exhibit a strong sexual dimorphism in which the sectioned with a glass or diamond knife. Thin sections were col- basitarsus of leg I is enlarged, spindle-shaped, lected using 1 3 2mm2 slot grids covered with formvar and and often fused (Kury, 1997; Townsend et al., stained sequentially in methanolic uranyl acetate and lead

Journal of Morphology 874 D.N. PROUD AND B.E. FELGENHAUER

Fig. 1. Scanning electron micrograph micrograph of the manaosbiid harvestman R. albilineatus, anteriolateral view.

citrate following Millonig (1976) for observation at 75 kV using ments of leg I (Fig. 2A–C), whereas females lack a Hitachi H-7600 transmission electron microscope. Semithin these structures (Fig. 2D). One pair of pores is sections of 300 lm were mounted on slides, stained with meth- ylene blue borax, and viewed and photographed using a Nikon located slightly distal to the center of the fused leg Eclipse E600FN with an attached Olympus DP71 camera. segments and one pair is located at the distal Specimens collected from Panama were exported under scien- region of these segments. tific permit number SE/A-11-07. Specimens from Trinidad were collected and exported under permit number 001339. Gland Structure RESULTS The swollen tarsal segments of leg I of the male External Leg Morphology R. albilineatus contain paired, multicellular, aci- Adult males of R. albilineatus (Fig. 1), C. montgo- nar glands (Fig. 3A) that exit through pores on the meryi,andB. williamsi possess a swollen, fused, ventral side of the leg (Fig. 2E,F; 5A). The two spindle-like basitarsus on leg I consisting of the basitarsal segments are fused (Fig. 2A–C), but two most proximal tarsomeres (Fig. 2A–C). This internally they possesses a separate pair of glands region of the leg is densely covered with sensilla each with a duct, termed here the conducting chaetica and trichomes in both males and females canal (Fig. 3A,C,E), and one glandular opening (Fig. 2A–D). The mean diameter of the swollen (Fig. 5A). The glands extend the entire length of basitarsus in males ranges from 390 lmfor the basitarsal segments. The proximal segment of C. montgomeryi to 550 lmforR. albilineatus (Table 1), the basitarsus contains much larger glands than and the thickness of the cuticle is approximately the distal segment, although all four glandular 30–35 lm. For all three species the male leg I structures possess similar ultrastructural aspects. basitarsal segments were significantly larger in The conducting canal, which appears to be epi- length and diameter than female leg I basitarsal cuticular, extends dorsally through the hypodermis segments (Table 1). Males and females of all three into the gland and runs proximally in the proximo- species examined exhibit a tarsal perforated organ distal axis along the length of the leg segment (Fig. 2G,H) at the most proximal region of the ven- (Figs. 3C, 5A). In the larger proximal gland, the tral side of the tarsus (Fig. 2A–D). Males possess a conducting canal is slightly shifted ventrally. Sev- pair of glandular openings (approximately 10–15 eral branches of the conducting canal (8) extend lm in diameter) on each of the two basitarsal seg- dorsally forming distinct lobes (Fig. 3C), the first

Journal of Morphology Fig. 2. Scanning electron micrograph showing external morphology of the sexually dimorphic leg I in male and female manaosbiid harvestmen. All micrographs are oriented with the proximal part of the leg to the right. A–D: Morphology of the sexually dimorphic leg I basitarsus showing the tarsal perforated organ (black arrows) at the proximal region, the numerous short trichomes (gray arrow), the less abundant longer sensilla chaetica (black arrow outlined in white), and the fused, spindleshaped basitarsal segments with paired glandular openings (white arrows) visible on each segment of the male leg. A: R. albilineatus, male, only three of the four glandular openings are visible, prolateral view. B: C. montgomeryi, male, ventral view. C: B. williamsi, male, ventral view. D: C. montgomeryi, female, note the lack of glandular openings, ventral view. E: C. montgomeryi, male, detailed view of glandular openings. F: R. albilineatus, male, detailed view of a glandular opening ﬁlled with a secretory product and surrounded by broken trichomes. G,H: Detailed view of the tarsal perforated organ located on the proximal, ventral surface of the most proximal tarsomere. G: B. williamsi,male.H:C. montgomeryi, male; inset shows high magniﬁcation of the small tarsal perforated organ pores. 876 D.N. PROUD AND B.E. FELGENHAUER

TABLE 1. Comparison of mean values (6SD) using Welch’s t test to show differences in length and diameter (in mm) of the basitarsus, comprised of the two most proximal tarsomeres, between males and females of the manaosbiids Rhopalocranaus albilineatus (10 males; 10 females), Barrona williamsi (3 males; 3 females), and Cranellus montgomeryi (15 males; 10 females)

Species Variable Males Females t df P

R. albilineatus Length 1.95 6 0.06 1.15 6 0.07 26.59 17.42 <0.001 Diameter 0.56 6 0.04 0.15 6 0.02 30.37 13.34 <0.001 B. williamsi Length 1.63 6 0.05 1.14 6 0.02 15.56 2.67 <0.01 Diameter 0.48 6 0.01 0.12 6 0.02 27.25 2.88 <0.001 C. montgomeryi Length 1.50 6 0.05 1.01 6 0.04 28.20 21.45 <0.001 Diameter 0.39 6 0.02 0.13 6 0.01 42.64 21.00 <0.001 of which is actually oriented in a dorsodistal direc- Additional Structures of Leg I tion. The smaller distal gland of the second tarso- Several other structures were observed in addi- mere has a conducting canal with only one branch- tion to the glands found in the male R. albilinea- ing duct that is oriented in a dorsodistal direction tus basitarsus of leg I. Just below the cuticle lies (Fig. 5A). the hypodermis (Fig. 3A). At the ventral side of The secretory cells of the gland are tall colum- the leg, running along the proximodistal axis, are nar cells that line the extracellular conducting structures that include tracheae, apodeme, and canal and empty their secretory products into other cells within the hemolymph (Fig. 3A). Open- small receiving canals at the apical region of the ings in the cuticle, which lead into the lumen of cell, which lead to the larger conducting canal sensilla chaetica, are also visible (Figs. 3E, 5B). (Fig. 3E,F). The secretory products may be of vari- ous sizes; large products (25–50 lm diameter) are stored in the apical region of the cell (i.e., adjacent Female Leg Structure to the cuticular canal), whereas many small products (<5 lm diameter) can be found throughout The female leg I of the three species examined the entire cytoplasm (Fig. 4A–D). The flattened, lacks the large glandular openings on the ventral elongate nucleus of the secretory cells is basally region of the leg (Fig. 2A). Examination of the located (i.e., adjacent to the hemocoel), ranges in female leg I of R. albilineatus using LM confirmed size from 15–20lm in length, and contains a large the absence of the large acinar glands (Fig. 3B,D). centrally located nucleolus (Fig. 4B). The secretory Like the male leg I, the female leg contains a cells are filled with large amounts of rough endo- tracheae, an apodeme, and the hypodermis plasmic reticulum (RER) and numerous mitochon- (Fig. 3B,D). The mean diameter of the female basi- dria (Fig. 4A–D). The well-developed RER in these tarsus ranges from 0.12 to 0.14 lm for all three secretory cells consists of multiplanar stacks of species (Table 1), and the thickness of the cuticle flattened lamellae and complex concentric or folded varies from 30 to 35 lm. lamellae. Two types of secretory products are present within the secretory cells of the glands and are DISCUSSION distinguishable based on differential metachro- matic staining using LM. One secretory product is The manaosbiid male basitarsus I demonstrates present in the majority of the secretory cells lining a significant increase in both tarsomere diameter the conducting canal, except in the region of the and tarsomere length when compared with females glandular openings. The second type of secretory for all three species. The tarsal perforated organ product was observed only in cells lining the found at the proximoventral region of the basitar- region of the conducting canal near the glandular sus was observed for males and females of all openings (Fig. 5A,B). The secretory products three species of manaosbiids examined. This struc- within these cells exhibit a granular substance ture occurs in this region on all four walking legs around the periphery (Fig. 5A,B). Further and is also common in Gonyleptidae (Willemart details regarding ultrastructure of the secretory et al., 2007) and Cosmetidae (unpublished data). cells near the glandular openings are currently The tarsal perforated organ function is unknown unknown. but it is probably of a glandular or sensory nature. Striated muscle reaches from the gland to The two large, paired glandular openings on the the inner wall of the cuticle on the dorsal side ventral region of the basitarsus are similar in of the proximal region of the gland (Fig. 5C). structure to the glandular openings described for The muscle attachment angle is 358 between the the manaosbiid I. strinatii for which only three muscle and cuticle. Cross section through the openings were observed (Willemart et al., 2010). muscle shows the individual actin and myosin In male R. albilineatus each of the four large filaments along with the presence of many mito- openings located on the first and second tarso- chondria (Fig. 5D). meres of the basitarsus serves as a pore through

Journal of Morphology Fig. 3. Light microscopy images of leg I of R. albilineatus comparing the internal morphology of male and female legs. A: Cross section through a male leg showing paired acinar glands with central conducting canals located within the hemocoel surrounded by the cuticle. Glandular cells containing secretory products and a large apically located nucleus are visible throughout each gland. Between the pair of glands is a bundle of striated muscle. Also visible are cells of the hypodermis, tracheae, and an apodeme. B: Cross section of female leg I showing cuticle, hypodermis, apodeme, and tissue-ﬁlled hemocoel. C: Sagittal section of the male gland showing the conducting canal running the length of the gland with branching ducts extending into lobes on the dorsal side of the gland. D: Sagittal section of the female leg showing the cuticle, hypodermis, and apodeme and lacking any large glands. E: Sagittal section of male leg showing a receiving canal (white arrowhead) through which the glandular cell empties secretory products into the conducting canal. The pore visible in the cuticle is associated with a sensillum chaeticum. F: Sagittal section of male leg showing secretory cells located laterally to the conducting canal. Several of the receiving canals (black arrowheads) that empty into the conducting canal are visible in cross section. ap, apodeme; bd, branching duct; cc, conducting canal; cu, cuticle; hy, hypodermis; lo, lobe; sm, striated muscle; n, nucleus; sc, pore associated with sensillum chaeticum; sp, secretory product; tr, trachea. 878 D.N. PROUD AND B.E. FELGENHAUER

Fig. 4. Transmission electron micrographs of the male leg I basitarsal glands in R albilineatus. A: Secretory cells contain an abundance of rough endoplasmic reticulum (RER) in complex concentric and folded lamellae at the apical region and along the periphery of the cell. B: Secretory cells contain a prominent nucleus, several large secretory products, and many small secretory products. C: RER is also seen as multiplanar stacks of flattened lamellae. This RER is located between two large secretory products with four small secretory products nearby. Inset shows high magnification of the ribosome studded RER indicated by the black arrow. D: An abundance of mitochondria are found throughout the cell amongst the small secretory products and multiplanar stacks of RER. m, mitochondria; n, nucleus; nu, nucleolus; rer, rough endoplasmic reticulum; spl, large secretory product; sps, small secretory product. which the products of a sexually dimorphic exo- the secretory cells found throughout the majority crine gland exit after passing through an epicuticu- of the gland (and not near the glandular openings). lar conducting canal. The two types of secretory Noirot and Quennedey (1974, 1991) developed a products observed are spatially organized within classification system to distinguish three different the gland. One product occurs throughout most of types of glandular cells in epidermal glands. Class the secretory cells in the gland and a second prod- III gland cells are usually part of a glandular unit uct, with a granular appearance around the consisting of one or more gland cells, which empty periphery, is restricted to secretory cells near the their products into a cuticular canal and a canal large glandular pores. These two secretory prod- cell that surrounds and constructs the conducting ucts can be distinguished by differential metachro- canal (Noirot and Quennedey, 1974, 1991). matic staining. It remains unknown whether In spiders, secretory organs commonly consist of ultrastructural differences occur between the secre- class III epidermal gland cells. Pheromone emit- tory cells producing these two products. We exam- ting glands comprised of class III secretory cells ined and described the ultrastructural aspects of associated with the female genital region have

Journal of Morphology SEXUALLY DIMORPHIC BASITARSAL GLANDS IN MANAOSBIIDS 879

Fig. 5. Light microscopy images and transmission electron micrographs of male leg I basitarsal glands and associated muscula- ture in R albilineatus. A: Sagittal section of the distal basitarsal gland showing the conducting canal exiting the leg via a large pore (approximately 10–15 lm in diameter) with a single branch in the dorsodistal direction and surrounded secretory cells containing a second type of secretory product. Receiving canals emptying into the conducting canal (white arrowheads) are also visible. B: Sagittal section showing the conducting canal in the region of the glandular opening and the two types of secretory products observed in the glandular cells. Several pores associated with sensilla chaetica are also visible. C: Sagittal section of the proximal basitarsal gland showing striated muscle attaching to the inner surface of the dorsal leg I cuticle. D: Cross section of striated muscle associated with the proximal basitarsal gland demonstrating the thin actin and thick myosin ﬁlaments and numerous mitochondria. bd, branching duct; cc, conducting canal; cu, cuticle; m, mitochondria; go, glandular opening; sm, striated muscle; sc, pore associated with sensillum chaeticum; sp, secretory product; sp2, second type of secretory product.

been described for several spider taxa (Kovoor, Taylor, 1977) and the femoral organ of zodariid 1981). For example, some female pholcids possess spiders (Peka´r and Sˆ obotnı´k, 2007). complex accessory glands in which the glandular Glandular units consisting of class III gland units consist of two gland cells and two enveloping cells are also described in several harvestmen taxa cells and are separated from other glandular units and display great complexity and diverse struc- by intercalary cells (Uhl, 1994). However, in some ture. For example, in the sexually dimorphic che- cases certain cells of the glandular units may liceral glands of nemastomatid harvestmen, the degenerate leaving only the secretory cell along glandular units are made up of six cells including with the cuticular canal (Noirot and Quennedy, three gland cells and three enveloping cells 1991). This type of class III gland cell occurs in (Martens, 1973). Martens (1979) showed that the the clypeal glands of linyphiid spiders (Blest and tarsal glands of male sironid harvestmen are made

Journal of Morphology 880 D.N. PROUD AND B.E. FELGENHAUER up of three gland cells, one enveloping cell, and whereas males commonly produce sex pheromones one duct cell. Juberthie et al. (1991) examined the that function at close proximity in courtship, ultrastructure of defense glands in ischyropsalid although rare cases of male attracting pheromones harvestmen and showed that glandular units con- have been reported (Gullan and Cranston, 2005). sist of at least eight gland cells, an intermediate Male sex pheromones in insects may function in cell, and several canal cells. male–male competitions; in signaling to a nearby According to the system of classification defined female the male’s identity, presence, and quality; by Noirot and Quennedey (1991), the gland cells or in stimulating a female into courtship behavior observed in the manaosbiid basitarsal gland are (Wyatt, 2003). However, pheromone-signaling roles class III gland cells. Although we were unable to involved in courtship behaviors in spiders are confirm the presence of canal cells during our ex- slightly different. Female spiders usually emit sex amination, the gland cells do empty their products pheromones (in the web silk or through cuticular through a cuticular canal. We believe that rela- glands) that may act as long-distance attractants tively small canal cells are probably present of males, signals of the female’s identity, and sex- around the cuticular canal and were simply not ual maturity and may also stimulate male court- observed. However, it is possible that the canal ship behaviors (Gaskett, 2007). cells are lost after formation of the canal similar to Our understanding of the reproductive biology of the glandular system observed by Peka´r and harvestmen is relatively limited, and courtship Sˆ obotnı´k (2007). behaviors, mating strategies, and parental care The presence of many mitochondria, a promi- differ greatly among families and even within gen- nent nucleolus, and an abundance of RER in these era (Machado and Macıás-Ordoń˜ ez, 2007). Given secretory cells are ultrastructural characteristics that the basitarsal glands are sexually dimorphic consistent with metabolically active cells that bio- and present only in males, it is possible that males synthesize proteins for export (Kuehnel, 2003). play the role of attracting, signaling, or courting a Striated muscle associated with the glands mate through the use of pheromones rather than attaches to the cuticle in the proximodorsal region females, as it often would be in spiders. Secretions and runs distally between the proximal glands. from sexually dimorphic glands used during court- The precise mechanism of expulsion of the secre- ship are known for several harvestmen species, for tory product is not completely understood, but example, the nuptial gifts provided to some dysp- these muscles possibly play an important role. noid females by the cheliceral glands of males In the clypeal glands of spiders, membrane (Martens, 1969). amplifications on the basal plasma membrane (e.g., Within the laniatorean lineage Grassatores, sev- microvilli or basal invaginations) and ER cisternae eral different families are known to exhibit packed along the basal and lateral plasma mem- enlarged tarsomeres on leg I (Pinto-da-Rocha and branes facilitate quick transport between hemo- Giribet, 2007; Townsend et al., 2010). Many lymph and the ER system, where part of the harvestmen of the families Cosmetidae (unpub. synthesis is thought to occur (Blest and Taylor, data; Willemart et al., 2010) and Gonyleptidae 1977). Similar amplifications on the basal plasma (Willemart et al., 2010) also possess sexually membrane and the packing of ER cisternae were dimorphic glandular openings on the enlarged, observed in the basitarsal glands of R. albilineatus. proximal tarsomeres of leg I in males. Our exami- From our examination, it appears that the secre- nation of the morphology and ultrastructure of the tory products are formed as small granules in the sexually dimorphic basitarsal glands in manaos- basal region of the cell and are merged to form biids provides a basis for which comparisons can larger granules in the apical region. A similar pat- be made between other manaosbiid species and tern was described for the dermal and defense other closely related families that exhibit swollen glands of some sironid harvestmen; following pro- tarsomeres. Gland structure and functional mor- duction of secretory granules, the small granules phology of such sexually dimorphic glands may are merged into larger granules before they are lead to comparative morphology-based studies and released by exocytosis into the secretory reservoir may help to improve our understanding of the (Gutjahr et al., 2005). relationships between certain laniatorean lineages. The function of the sexually dimorphic basitarsal glands possessed by male manaosbiid harvest- ACKNOWLEDGMENTS men is unclear. Based on the better-understood ev- olutionary patterns for the reproductive biology of We thank V. Townsend for lending specimens insects and spiders, we believe the manaosbiid from Panama and assisting with field collections basitarsal glands secrete a sex pheromone that in Trinidad; T. Pesacreta and M. Purpera of the plays an important role in their reproductive University of Louisiana at Lafayette (ULL) Micros- behaviors. copy Center, who provided access to and assistance In many insects, females often produce sex pher- with microscopy and imaging; S. Broadbridge for omones that attract males over long distances, his assistance with transportation and logistics in

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