2014. The Journal ofArachnology 42:248-256 Chemical defenses in the opilionid infraorder Insidiatores: divergence in chemical defenses between Triaenonychidae and Travunioidea and within travunioid harvestmen (Opiliones) from eastern and western North America W. A. Shear', T. H. Jones-, H, M. Guidry-, S. Derkarabetian^"', C. H. Richart"*"*, M. Minor^ and J. J. Lewis^: 'Department of Biology, Hampden-Sydney College, Hampden-Sydney, VA 23943, USA. E-mail; [email protected]; -Department of Chemistry, Virginia Military Institute, Lexington, VA 24450, USA; ^Department of Biology, San Diego State University, San Diego, CA 92182, USA; ''Department of Biology, University of California, Riverside, Riverside, CA 92521, USA; ^Ecology Group, Institute of Agriculture & Environment, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand; '’J. Lewis & Associates Biological Consulting, 217 W. Carter Avenue, Clarksville, IN 47129, USA Abstract. Livewhole specimens oftwo species ofthe harvestman Stiperfamily Travunioidea Absolon & Kratchovil 1932 from the eastern United States, eight species from the western United States, six morphospecies of the family Triaenonychidae Sorensen 1886fromNewZealand,and specimensofthephylogeneticallyearly-divergingNorthAmerican triaenonychid Fwmmtaiia dejvchoidor Shear 1977 were extracted in methanol, and the solvent analyzed for components from their'defensive secretions. The components were then mapped on a recent phylogeny of the taxa. In both eastern cladonychiid species, Erehonuister flavescens Cope 1872 and Theronuister bnmneus (Banks 1902), the major component found was anabaseine, an alkaloid related to nicotine. In the western species, Parcmonyclms hrunneus (Banks 1893), Cryptonuister leviathan Briggs 1969, Speleoniaster lexi Briggs 1974, S. pecki Briggs 1974, Speleonychia sengeri Briggs 1974, Metanonychiis idahoensis Briggs 1971, Briggsus flavescens (Briggs 1971) and Sclerohiinus nondiniorphicus Briggs 1971, the major component was N,N-dimethylphenylethylamine, implying that the travunioids from the two regions represent different phyletic lines. The secretions ofthe triaenonychid species, members ofthe genera Soerensenella Pocock 1903 and Nnncia Loman 1902, were dominated by 4-methyl-3-hexanone, and that of F. depreliendor by phenol. The completely different chemistry ofthe two taxa, Travunioidea and Triaenonychidae, implies significant phylogenetic differences, and the presence ofphenol in F. depreliendor may suggest a long period ofseparate evolution for this species. Keywords: Nicotine,benzothiazole,2-3'dipyridyl,salicylalcohol,mellein,N,N-dimethylphenylethylamine,4-methyl-3-hexanone Harvestmen, arachnids ofthe orderOpiliones(also known in While these studies focused primarily on reporting the North America as daddy-long-legs) defend themselves chemi- composition ofsecretions from individual species, some recent cally with secretions from paired glands in the prosoma, which work has been more analytical. Rocha et al. (2013) discussed open through pores on either side ofthe body. Information on possible chemical pathways for the synthesis of secretion the chemical composition of these secretions has accumulated components. Attempts at a phylogenetic analysis of the since the initial studies of Estable et al. (1955) that identified distribution of defensive secretions include those of Caetano gonyleptidine, the first defensive substance from a harvestman & Machado (2013) and Raspotnig et al. (2014). The hope has to be chemically determined. Developments in the field have frequently been expressed that data on defensive secretions been ably summarized in a chapter by Gnaspini & Hara (2007), may be of value in the phylogenetics and taxonomy of which revealedthat research ondefensivechemistryin Opiliones Opiliones (Hara et al. 2005; Jones et al. 2009; Shear et al. has focused disproportionally on South American gonyleptids 2010a, b, Fdttingeret al. 2010, Raspotnig 2012 [2013]), but we and their relatives(seealso Ebttingeret al. 2010). Since the2007 see an emerging picture that may be blurred by a great deal of review, information has been added regarding more disparate homoplasy. Indeed, the results ofthe analyses ofthe samedata taxa for which the chemistry of the secretions was previously by Caetano & Machado (2013) and Raspotnig et al. (2014) unknown. Raspotnig et al. (2005) published the first report on came to opposite conclusions concerning the polarity of the chemistry of sironids (Cyphophthalmi Simon 1879), and chemical transformations in Grassatores. Jones et al. (2009) added data for a stylocellid. Raspotnig et al. Traditional Opiliones taxonomic groups have now been (2010) provided the first report of secretion chemistry among robustly supported with genomic data sets (Hedin et al. 2012), Dyspnoi, from Panineimistoma quadripiinctatum (Perty 1833), and include the mite-like suborder Cyphophthalmi as sister to and Shear et al. (2010a, b) studied two North American remaining harvestmen, the Phalangida Latrielle 1796. Within phalangodids, Bishopella laciniosa (Crosby & Bishop 1924) and Phalangida, the raptorially-pedipalped Laniatores Thorell Texella hifiircata (Briggs 1968), and a stygnopsid, Cltiiuptepello- 1876 are sister to the Palpatores Thorell 1876, comprised of hiiinis nuuHae (Goodnight & Goodnight 1967). These more the often long-legged suborder Eupnoi Hansen & Sorensen recent developments havebeen summarized by Raspotnig (2012 1904 and the suborder Dyspnoi Hansen & Sorensen 1904. The [2013]), who also mentioned preliminary results for many division of the suborder Laniatores into two infraorders, additional harvestman species. Thus while progress has been Insidiatores Loman 1900 and Grassatores Kury 2003, was made filling taxonomic gaps in our knowledge of harvestman proposed by Kury (2003) to taxonomically recognize two defensive secretions, much remains to be done. divergent phyletic lines of harvestmen. Insidiatores includes 248 SHEAR ET AL.—CHEMICAL DEFENSE OF INSIDIATORES 249 those taxa presently grouped as Triaenonychidae Sorensen placed asvouchers in the collection ofthe Virginia Museum of 1886, Synthetonychiidae Forster 1954, and a group ofspecies Natural History, Martinsville, Virginia. of unsettled family-level taxonomy presently referred to as Although when it was possible to extract more than one Travunioidea Absalon & Kratchovil 1932. It is not clear that specimen ofa species separately, the results were consistent, in Insidiatores as composed is monophyletic (but Grassatores most cases wewere restricted to a single specimen by the rarity almost certainly is). Representative Insidiatoresexamined here of the species involved and the difficulties in collecting them, can be seen in Fig. 1. or analyzed extracts from several specimens collected into the Synthetonychiidae is a poorly studied but probably same vial. For this reason, some of our results must be monophyletic taxon including minute harvestmen limited to regarded as preliminary, and we are working to follow up with New Zealand (Forster 1954, Kury 2007). In some recent additional specimens. However, at the level we are studying, phylogenies, synthetonychiids have been resolved as an simply characterizing components without detailed quantita- outgroup to the remaining Laniatores (Giribet et al. 2010). tive analysis, previous studies have shown little variation Triaenonychidae is composed ofnumerous genera and species within species in the composition of their secretions, though that are important, if not dominant, elements of the relative amounts ofcomponents may differ. HMG harvestman fauna of the southern hemisphere (Australia, The analysis of the extracts was performed by and New Zealand, Madagascar, South Africa, and southern South THJ. Gaschromatography-mass spectrometry was carried out America [Kury 2007]), but one species, Fumontami deprehen- in the El mode using a Shimadzu QP-5000 or QP-2010 GC/ dor Shear 1977, is known from the southern Appalachian MS equipped with an RTX-5, 30 m X 0.25-mm i.d. column. Mountains in North America (Shear 1977, Thomas & Hedin The instruments were programmed from 60 ° C to 250 ° C at 2008). Triaenonychid taxonomy is somewhat problematical 10 7min. Identification of components was accomplished (Mendes& Kury2008). No triaenonychids had been examined using NIST/EPA/NIH mass spectral library on CD-rom, for the chemistry of their defensive secretions prior to this version 1.7 (1999) and the NIST/EPA/NIH mass spectral study, and synthetonychiids remain unstudied. library version 2.0d (2005). Genera and species ofthe “superfamily” Travunioidea have All chemicals were mapped onto a modified phylogeny been recorded from Europe (Kury& Mendes 2007) and Japan, based on the molecular phylogenetic analysis of Derkarabe- but North America appears to host the most diverse and tian et al. (2010), trimmed to include only those genera with probably the best understood fauna (Fig. 1; Shear & chemical data presented here. An ultrametric tree was used Derkarabetian 2008, Derkarabetian et al. 2010, 2011). Only for character mapping, which was conducted in Mesquite a single North American species from this phylogenetically 2.75 using the ancestral state reconstruction module using important taxon has been examined from the viewpoint of parsimony. Additionally, we mapped chemicals onto a chemical defense. Specimens from New Mexico were studied phylogeny including triaenonychids analyzed here and the by Epka et al. (1984); at the time they referred their material genus Holoscotolemon. The taxa were added according to to Sclerobumis rohustiis (Packard 1877), but recent work their placement in the maximum likelihood phylogeny of (Derkarabetian et al. 2010, 2011; Derkarabetian & Hedin Giribet et al. (2010). 2014) has shown that at least three additional species occur in RESULTS New Mexico, so the exact identity of their specimens is now unclear. Epka et al. (1984) found an extraordinary array of Results of the analysis are presented in Tables 2M, and molecules in the secretion of S. robustus: N,N-dimethylphe- structural formulae of detected components are shown in nylethylamine, nicotine, bornyl acetate, bornyl propionate, Fig. 2. As seen in Table 2, the major component of the camphene and limonene. secretion in both eastern North American travunioid species Raspotnig et al. (2011) examined four species in the {ErebomasterJlavescens Cope 1872 and Theromaster briinneus European travunioid genus Holoscotolemon Roewer 1915; (Banks 1902)) was the alkaloid anabaseine. Minor or trace H. jaqueli (Corti 1905), H. oreophUum Martens 1978, H. components were anabasine (a related alkaloid), phenol, lessiniense Martens 1978 and H. imicolor Roewer 1915. They benzothiazole, salicyl alcohol, 2,3'-dipyridyl and mellein. found that the secretions ofH.jacpwtiand H. oreophUum were Four individuals of T. brunneus were analyzed; no significant dominated by nicotine, while that of H. lessiniense primarily differences were found between individuals, except that salicyl consisted of the similar alkaloid anabaseine. No results were alcohol was not found in two ofthe specimens. A specimen of obtained from adults ofH. unicolor. E. Jlavescens from Indiana was analyzed separately, and six For this study, we analyzed extracts from 15 species of specimens of the species from Ohio were extracted and Insidiatores from North America and New Zealand. While analyzed as a group. The results for E. jlavescens differed our findings for the North American species might have been from those for T. brunneus in that trace amounts of 4- predicted from the earlier examinations of Sclerohunus hydroxybenzine-ethanol were found in the E. Jlavescens ?robustus and the European species of Holoscotolemon, the extract, and that phenol, anabasine and mellein were minor chemistry ofthe New Zealand forms was quite unexpected. components (1-10%) rather than traces (< 1%). METHODS Table 3 summarizes the results from the analyses ofextracts from eight species of travunioids from western North Specimens studied were collected alive and dropped in the America. Components in common with the eastern species field into vials containing less than 1 ml of USP methanol; were phenol and benzothiazole, and as with the eastern the vials had Teflon-lined caps. Collection localities for the species, these compounds were present only in trace amounts. specimens studied are given in Table 1. All specimens will be The major component in all western species was N,N- 250 THE JOURNAL OF ARACHNOLOGY mm 1 mm 0.5 mm mm 1 1 mm 1 mm 1 mm 1 mm mm 0.5 1 Figure 1. Representatives of North American Insidiatores. High resolution images for all specimens figured here are available on Morphbank under publication ID 835667 {http://www.morphbank.net/835667). A. Briggsiis flavesceiis, B. Cryptomaster leviatium, C. Paranoiiychus hriinneiis, D. Metanoiiycinis ulcilioeitsis, E. Fitmontaiia deprehendor, F. Erehomaster sp., G. Speleomaster lexi, H. Speleomaster pecki, I. Speleoiiychia sengeri. SHEAR ET AL.—CHEMICAL DEFENSE OF INSIDIATORES 251 — Table 1. Collecting localities. Voucher Species number Collection localities Cryptomaster leviathan 07-177 OR: Lane Co., Willamette Nat. For., Clark Creek Organization Camp, 28 May 2007, A.Richart, C.Richart (CHR 1354) 0078--188 OR: Coos Co., Golden and Silver Falls St. Pk., 4 April 2008, S.Derkarabetian, C.Richart (CHR 2029) 07-176 OR: Lane Co., Willamette Nat. For., Clark Creek Organization Camp, 28 May 2007, A.Richart, C.Richart (CHR 1335) Erebomaster flavescens 000877---117890 IINN::CHraarwrfiosrodnCCoo..,,SDibeevritl’ssWGerlalveCyaavred(nCeaavreW,y7anmdiotStWeCCaovre)y,do3nm,i1N9ENoLevav2e0n0w7o,rtJ.h,Le1w9iNsov2007,J. Lewis 181 IN: Harrison Co., Devils Graveyard Cave, 7 mi SW Corydon, 19 Nov 2007, J. Lewis 12-336 OH: Adams Co., Edge ofAppalachia Preserve, 8 June 2011, W. A. Shear Theromaster bnmneus 08-211 NC: Haywood Co., Cullowhee Mtn. Road at WolfCreek, 22 October 2008, W. A. Shear Speleomaster lexi 08-172 ID: Lincoln Co., Tee Cave, 30 June 2007, A.Richart, C.Richart (CHR 1577) 178 ID: Lincoln Co., Gwinn Cave, 29 June 2007, A.Richart, C.Richart (CHR 1568) Speleomasterpecki 08-174 ID: Butte Co., Beauty Cave, 30 June 2007, A.Richart, C.Richart (CHR 1581) Speleonychia sengeri 08-175 WA: Klickitat Co., Cheese Cave, 9 June 2007, N.Richart, C.Richart (CHR 1621) 08-176 WA: Skamania Co., Cave #27, 9 June 2007, N.Richart, C.Richart (CHR 1622) 08-177 WA: Skamania Co., Big Cave, 8 June 2007, N.Richart, C.Richart (CHR 1588) 08-179 WA: Skamania Co., Slime Cave (Cave #39) 8 June 2007, N.Richart, C.Richart (CHR 1607) Paranonychus bnmneus 07-174 OR: Lane Co., Willamette Nat. For., Clark Creek Organization Camp, 28 May 2007, A.Richart, C.Richart (CHR 1356) 07-175 OR: Lane Co., Willamette Nat. For., Clark Creek Organization Camp, 28 May 2007, A.Richart, C.Richart (CHR 1357) Metanonyclms idahoensis 09-248 ID: Shoshone Co., Hobo Cedar Grove, 25 July 2008, C.Richart (CHR 2361) Briggsus flavescens 08-190 OR: Clatsop Co., Saddle Mt. Rd. near U.S. 26, 3 April 2008, S.Derkarabetian, C.Richart (CHR 2016) Nimcia sp. 10-275 NZ: South Island, Westland, Dancing Creek, Haast Pass, 11 February 2010, M. Minor Nimcia sp. 10-278 NZ: South Island, Buller, Aratika, 9 February 2010, M. Minor Nimcia sp. 10-279 NZ: South Island, Buller, Springs Junction, 5 February 2010, M. Minor Soerensenella sp. 10-271 NZ: North Island, Wanganui, Totara Reserve, 28 March 2010, M. Minor Soerensenellaprehensor 10-272 NZ: North Island, Taupo, Whakapapa Bush, 4 April 2010, M. Minor dimethylphenylethylamine, with nicotine and N,N-dimethyli- either one or two specimens. The major components of the soamylamine as minor or trace components. An exception New Zealand triaenonychoids were quite different from both was Briggsus flavescens (Briggs 1971), in which the major F. deprehendor and the travunioids. While the travunioids and component was phenol, with N,N-dimethylphenylethyIamine F. deprehendor were dominated by cyclic compounds fre- as a minor component and a trace amount of benzothiazole. quently containing nitrogen, the New Zealand triaenony- This unexpected result came from one small specimen and choids showed linear aldehydes, alcohols and ketones. The requires confirmation. secretions were also much less complex, with only one or two Table 4 shows results from the analyses of extracts of minor or trace components in Nimcia sp. triaenonychids. Three small specimens ofF. deprehendor were Results of the character mapping analyses including the extracted and analyzed together. Fumontana deprehendor had triaenonychids and Holoscotolemon are shown in Fig. 3. This phenol as a major component, with traces of salicyl alcohol. analysis indicates that if Insidiatores is monophyletic, the Each record of a New Zealand triaenonychoid represents ancestral state for all species is phenol, with changes to 4- — Table 2. Compounds present in eastern North American travunioids and species of Holoscotolemon (data on Holoscotolemon from Raspotnig et al. 2011). Plus sign indicates major component, “o” a minor component (<10%) and “t” a trace component (<1%). The “Unknown” is an undetermined component at m/z = 174. Erebomaster Theromaster Holoscotolemon Holoscotolemon Holoscotolemon Fig. 2 Component flavescens bnmneus jacpietf lessiniense‘ oreophilum’ 1 Phenol o t 2 Benzothiazole t t 3 Salycyl alcohol t t 4 4-Hydroxybenzenethanol t 5 Anabasine o t 6 2,3'-Dipyridyl t t t 7 Anabaseine + + + 8 Mellein 0 t 10 Nicotine + + Unknown* t THE JOURNAL OF ARACHNOLOGY 252 (<1%). o a. + ^ O o o component trace a + o ^ “t” c oa + + and S o o (<10%) + o o component minor Cl, a “o” component, + ’O c c3 major N za> indicates + sign o Plus -C p, o p: + travunioids. ’2 American North ^ § western <u in ^<^U sOCl 2 5= present -Q. so O X Cl ^ O (U OJ oa x: 'CcO/5 1o) o^ _ . Compounds U£5 :oa3 i^: "co o "S •^£ ’-<i£.u '-aSL> o u r ^ S e- oo t ^ — Oh CQ Z Z Z I. a 3. — ri O PO q' IT) Table SHEAR ET AL.—CHEMICAL DEFENSE OF INSIDIATORES 253 ship of the eastern North American genera with European Holoscotolemon than with the travunioid genera from western North America. For the western travunioids, N,N-dimethylphenylethyla- mine was the major component in all species except Briggsus flavescens. Metanonychus klahoensis Briggs 1971 and Scler- ohunus nondimorphiciis Briggs 1971 had nicotine and N,N- diethylisoamylamine as minor components, as well as two unidentified compounds not shown. For the other species, phenol was present as either a minor component or a trace, and benzothiazole was found as a trace in Parammyclnis brunneus (Banks 1893) and Speleonychia .sengeri Briggs 1974. The complex mixtures found in the eastern cladonychiids and in S. ?robustus (Epka et al. 1984) were not recovered from the western species we studied. The complexity of the secretion extracted from the two eastern cladonychiid species is similar to that found by Epka et al. (1984) for Sclerobimus ?robustus, but quite different chemically. New Mexico Sclerobimus Banks 1893 require re-examination. Both the complexity and the diversity of chemical compo- sition within Insidiatores is unusual among opilionids, because in previous studies, similar classes of compounds (though different molecules) have been found in large taxonomic groupings. For example, sclerosomatids utilize a variety of ketones and alcohols, and many Grassatores produce alkyl- phenols and hydroquinones (Hara et al. 2007, Raspotnig 2012 [2013], Gaetano & Machado 2013, Raspotnig et al. 2014). In some cases the secretion consists ofa single compound (Shear et al. 2010a, b). However, in the case ofthe cyphophthalmids, the two species so far studied show as diverse an array of molecules as do the travunioids or even more so (Raspotnig et 14 15 al. 2005, Jones et al. 2009, Raspotnig 2012 [2013]). Because — Figure 2. Compounds identified in this study. I. Phenol, 2. cyphophthalmids are sister to all remaining Opiliones, the Benzothiazole, 3. Salycyl alcohol, 4. 4-Hydroxybenzenethanol, 5. scanty data collected so far could be construed to suggest that Anabasine, 6. 2,3'-dipyridryl, 7. Anabaseine, 8. Mellein, 9. N,N- early-evolving defensive secretions were complex mixtures, dimethylphenylethylamine, 10. Nicotine, 11. N,N-dimethylisoaniyla- later winnowed down to only a few, or to single, components. mine. 12. 4-methyl-3-hexanone, 13. Methylhexanoate, 14. 4-methyl-3- Evidence against this view is that gonyleptoids, a derived hexanol, 15. 4-methyl-3-heptanone. group, also have complex mixtures, though the compounds are nearly all methylated and/or ethylated benzoquinones or methyl-3-hexanone in New Zealand triaenonychids and to alkylphenols (Fottinger et al. 2010, Raspotnig 2012 [2013]). N,N-dimethylphenylethylamine in travunioids. However, the question that remains unexamined so far is the extent to which the method ofcollecting the secretions and the DISCUSSION processing for analysis may have influenced the results; it is The qualitative near-identity of the extracts from E. possible that chemical changes in some of the components flavescens and T. brunneus supports the close phylogenetic could be induced during study, and this could account for the relationship hypothesized on the bases of morphology and mixtures obtained. genetics by Derkarabetian et al. (2010). The strong differences Results of the character mapping for Travunioidea are between the secretions ofthis “eastern clade” and that of the shown in Fig. 4. The various compounds are represented by hypothetical “western clade” of travunioids supports that numbers that correspond to those in Fig. 2. Two major distinction. findings are seen in the parsimony reconstruction regarding Raspotnig et al. (2011) found anabaseine as the major the chemicals that constitute the major components. First, the component in the secretion ofHoloscotolemon lessiniense, but major component N,N-dimethylphenylethylamine (9) was nicotine dominated that of H. jaqueti and H. oreopliilimi recovered as the ancestral state for all travunioid genera (Table 2). These three species appearto beclosely related from included in this analysis. Second, there is a transition from morphological evidence and numerous characters, especially N,N-dimethylphenylethylamine (9) to anabaseine (7) as the genitalic, place them close to Erehomaster Cope 1872 and major component on the branch leading to the eastern Theromaster Briggs 1969 (Martens 1978). Trace components Cladonychiidae (Erebonuister and Theromaster). In addition, in these three species were pyridines with the same core these two genera also possess many other minor or trace structure as anabaseine and nicotine. Both chemical and elementsthat are unique to this lineage, namely salycyl alcohol morphological evidence, therefore, argue for a closer relation- (3), anabasine (5), 2,3'-dipyridyl (6) and mellein (8). Also, 254 THE JOURNAL OF ARACHNOLOGY Phenol 2. Benzothiazole rnFumontana Fumontana sSoerensenellaprehensor Soerensenellaprehensor aSoerensenellasp. Soerensenellasp. aNuncia sp. SpringsJunction 1 Nunciasp. Springs Junction 1 aNuncia sp. SpringsJunction 2 Nunciasp. SpringsJunction 2 uNuncia sp. DancingCreek Nunciasp. DancingCreek sNuncia sp. Aratika Nuncia sp. Aratika tCryptomaster leviathan Cryptomaster leviathan rnSpeleomasterpecki Speleomasterpecki mSpeleomasterlexi Speleomasterlexi rnErebomasterflavescens Erebomasterflavescens tTheromaster brunneus Theromaster brunneus aHoloscotolemon lessiniense Holoscotolemon lessiniense aHoIoscotolemon oreophilum Holoscotolemon oreophilum aHoloscotolemonjaqueti Holoscotolemonjaqueti mBrigssusflavescens Brigssusflavescens tSpeleonychiasengeri Speleonychiasengeri aParanonychus brnnneiis Paranonychus brunneus aMetanonychus idahoensis Metanonychus idahoensis aSclerobunus nondimorphicus Sclerobunus nondimorphicus 6,7. 2,3’Dipyridyl andAnabaseine Fumontana rnFumontana Soerensenellaprehensor aSoerensenellaprehensor Soerensenellasp. sp. Nuncia sp. Springs Junction 1 uNuncia sp. Springs Junction 1 Nuncia sp. Springs Junction 2 aNuncia sp. Springs Junction2 Nuncia sp. Dancing Creek uNuncia sp. Dancing Creek Nuncia sp. Aratika uNuncia sp. Aratika Cryptomaster leviathan 3Cryptomasterleviathan Speleornasterpecki aSpeleomasterpecki Speleomasterlexi ttSpeleomasterlexi Erebomasterflavescens rnErebomasterflavescens Theromaster brunneus aTheromaster brunneus Holoscotolemon lessiniense rnHoloscotolemon lessiniense Holoscotolemon oreophilum aHoloscotolemon oreophilum Holoscotolemonjaqueti uHoloscotolemonjaqueti Brigssusflavescens aBrigssusflavescens Speleonychiasengeri c aSpeleonychiasengeri Paranonychus brunneus aParanonychus brunneus Metanonychus idahoensis aMetanonychus idahoensis Sclerobunus nondimorphicus aSclerobunus nondimorphicus 9. N,N-dimethylphenylethylamine 10. Nicotine sFumontana uFumontana ^Soerensenellaprehensor aSoerensenellaprehensor ^Soerensenellasp. aSoerensenellasp. sNuncia sp. SpringsJunction 1 aNuncia sp. Springs Junction 1 sNuncia sp. SpringsJunction 2 aNuncia sp. Springs Junction 2 ^Nuncia sp. Dancing Creek aNunciasp. Dancing Creek I[ Nuncia sp. Aratika aNuncia sp. Aratika Cryptomaster leviathan Cryptomaster leviathan 1 Speleomasterpecki aSpeleomasterpecki Speleomasterlexi aSpeleomasterlexi Erebomasterflavescens nErebomasterflavescens Theromaster brunneus iTheromasterbrunneus Holoscotolemon lessiniense aHoloscotolemon lessiniense ^Holoscotolemon oreophilum rnHoloscotolemon oreophilum [I nHoloscotolemonjaqueti aHoloscotolemonjaqueti I l^-am^mBrigssusflavescens aBrigssusflavescens ’’“tmm^.mSpeleonychiasengeri aSpeleonychiasengeri aParanonychus brunneus aParanonychus brunneus mMetanonychus idahoensis aMetanonychus idahoensis aSclerobunus nondimorphicus aSclerobunus nondimorphicus — Figure 3. Results ofchemical character mapping for Insidiatores. Only those chemicals with 2 or more steps are shown. Black = presence, white = absence. SHEAR ET AL.—CHEMICAL DEFENSE OF INSIDIATORES 255 disrupt the characterizations given. Among the anomalies Raspotnig (2012 [2013]) mentions which require explanation are the presence of naphthoquinones in some putative sclerosomatids (Gyas sp.), ketones in some Gonyleptidae (Grassatores), and now, as a result of our work, methyl ketones (“sclerosomatid substances”) in Triaenonychidae and phenol in Fumontanadeprehendor, a species thatconsistently is recovered in phylogenies as sister to remaining triaenonychids. At least these lattertwo make possible a tentative link between Laniatores and some Palpatores. Caetano & Machado (2013) conducted a phylogenetic analysis of the distribution of scent gland chemistry in Grassatores, and concluded that benzoquinones were ances- nioidea. Numbers correspond to the chemicals listed in Tables 1 and tral, with alkylphenols evolving independently many times. 2. Boxednumbersaboveabrancharecharactergains,thosebeloware Using the same data, but a different method ofanalysis and a losses. Bold boxes are major components and regular boxes are different—outgroup, Raspotnig et al. (2014) concluded the minor/trace components. Dashed boxes represent those chemicals opposite that benzoquinones were derived and alkylphenols that are equally parsimonious (present/absent) along the branch; but ancestral. Based on the methods used and the fact that branches with definite gains for these chemicals are also included. Raspotnig et al. (2014) used a more appropriate outgroup, we Boxes with asterisks indicate a change in component concentration agree with the latter conclusion. Our finding that phenol is (e.g., change from major to minor). probably ancestral in Insidiatores (see Fig. 3) reinforces this, although exact phylogenetic relationships between Insidiatores Erehomaster is the only taxon known to possess 4-hydro- and Grassatores remain unclear. xybenzenethanol (4). The sclerobunines (Sclerobiiuus and Raspotnig (2012 [2013]) did not attempt to map the known Metanonychus Briggs 1971) have lost phenol (1) as a characters on any established phylogenetic tree of Opiliones. component but have gained both nicotine (10) and N,N- However, study ofhis Table 2 (pp. 9-10) and our Fig. 3 seems dimethylisoamylamine (11). Interestingly, two species of to indicate that at least at the present state ofknowledge, there Holoscotolemon also produce nicotine. is a great deal of homoplasy present, with various types of Raspotnig (2012 [2013]) discussed at length the possible compounds being lost and then regained, or evolving phylogenetic and systematic implications of the diversity of independently. defensive compounds in Opiliones. Overlooking some disso- In our results for Insidiatores, the most divergent observa- nant results, it appears that the suborder Cyphophythalmi can tion is the presence of 4-methyl-3-hexanone as the major be characterized by methyl ketones, naphthoquinones and component in all of the New Zealand triaenonychids we related compounds. Benzoquinones appear in phalangiid studied. If we consider Fumontana as a plesiomorphic Eupnoi, and “sclerosomatid compounds" (noncyclic ketones, outgroup, we have the problem of getting from phenol to alcohols and aldehydes, such as 4-methyl-3-hexanone) are these noncyclic ketones. The travunioids stand alone with the found in sclerosomatid Eupnoi. Few Dyspnoi have been predominant secretion of either N,N-dimethylphenylethyla- examined, but naphthoquinones and anthraquinones have mine or tobacco alkaloids like nicotine and anabaseine. A been found. Grassatores produce predominantly phenols, major question, which by extension could be applied to the benzoquinones and hydroquinones. Insidiatores, up to the entire phylogenetic scheme of this character, is how one gets findings of this study, were characterized by nitrogen- from one compound or set of compounds in a supposed containing alkaloids. Raspotnig (2012 [2013]) is quick to plesiomorphic taxon to a chemically completely different point out that taxonomic sampling within the Opiliones has compound further up in the tree. In other words, is it been erratic and many taxa remain unsampled, or known only reasonable to assume a transition from phenol to 4-methyl-3- from unpublished or preliminary results. hexanone? Raspotnig (2012 [2013]) proposed a number ofphylogenetic ACKNOWLEDGMENTS hypotheses that may be summarized as follows: 1) complex mixtures ofsecretions are plesiomorphic compared to uniform Analysis facilities were provided by the Department of WAS or less diverse mixtures; 2) naphthoquinones and methyl Chemistry at Virginia Military Institute. thanks Dr. ketones, as found in cyphophthalmids, are basal; 3) naphtho- Fred Coyle for hospitality and guidance in western North quinones are synapomorphic for a clade Cyphophthalmi + Carolina, and Chris Bedel and the staff of the Edge of Palpatores; 4) acyclic compounds in Cyphophthalmi and Appalachia Preserve, West Union, Ohio. Participation of Sclerosomatidae may have a common origin; 5) “sclerosoma- WAS was supported by a grant from the Professional tid compounds” may represent a synapomorphy for Palpa- Development Committee of Hampden-Sydney College. Field- tores; 6) a deep chemical divergence separates Insidiatores and work in western North America was supported by grants from Grassatores; and 7) a link between the chemistry of the American Arachnological Society Vincent Roth Fund for Cyphophthalmi -i- Palpatores and Laniatores remains to be Systematic Research. Adrienne Richart, Nicholas Richart, found. and William P. Leonard helped secure specimens. Alexa But the phylogenetic signal is not so clear as that. The R. Feist imaged specimens and accessioned images to dissonant results mentioned above seem to significantly MorphBank. 256 THE JOURNAL OF ARACHNOLOGY LITERATURE CITED da-Rocha, G. Machado & G. Giribet, eds.). Harvard University Caetano, D. & G. Machado. 2013. The ecological tale ofGonylepti- Press, Cambridge, Massachusetts. dae (Arachnida, Opiliones) evolution: phylogeny ofa Neotropical Kury, A.B. & A. Cruz Mendes. 2007. 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