Light from dark: A relictual troglobite reveals a broader ancestral distribution for kimulid harvestmen (Opiliones: Laniatores: Kimulidae) in South America


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Light from dark: A relictual troglobite reveals a broader ancestral distribution for kimulid harvestmen (Opiliones: Laniatores: Kimulidae) in South America

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Light from dark: A relictual troglobite reveals a broader ancestral distribution for kimulid harvestmen (Opiliones: Laniatores: Kimulidae) in South America
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PLoS ONE
Creator:
Pérez-González, Abel
Ceccarelli, F. Sara
Monte, Bruno G. O.
Proud, Daniel N.
Bernardino DaSilva, Márcio
Bichuette, Maria E.
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Relictual Troglobite ( local )
Ancestral Distribution ( local )
Kimulid Harvestmen ( local )
Opiliones: Laniatores: Kimulidae ( local )
South America ( local )
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serial ( sobekcm )

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A new troglobitic harvestman, Relictopiolus galadriel gen. nov et sp. nov., is described from Olhos d’Água cave, Itacarambi, Minas Gerais State, Brazil. Morphological characters, including male genitalia and exomorphology, suggest that this species belongs to the family Kimulidae, and it appears to share the greatest similarities with Tegipiolus pachypus. Bayesian inference analyses of a molecular dataset strongly support the inclusion of this species in Kimulidae and confirm the hypothesized sister-group relationship between R. galadriel and T. pachypus. A time calibrated phylogeny indicates that these sister-taxa diverged from a common ancestor approximately 40 Mya, during the Paleogene. The current range of Kimulidae illustrates a remarkable disjunct distribution, and leads us to hypothesize that the ancestral distribution of Kimulidae was once much more widespread across eastern Brazil. This may be attributed to the Eocene radiation associated with the warming (and humidifying) events in the Cenozoic when the best conditions for evergreen tropical vegetation in South America were established and followed by the extinction of kimulid epigean populations together with the retraction of rain forests during the Oligocene to Miocene cooling. The discovery of this relictual troglobite indicates that the Olhos d’Água cave was a stable refugium for this ancient lineage of kimulids and acted as a "museum" of biodiversity. Our findings, considered collectively with the diverse troglofauna of the Olhos d’Água cave, highlight it as one of the most important hotspots of troglobite diversity and endemism in the Neotropics. Given the ecological stresses on this habitat, the cavernicolous fauna are at risk of extinction and we emphasize the urgent need for appropriate conservation actions. Finally, we propose the transfer of Acanthominua, Euminua, Euminuoides and Pseudominua from Kimulidae to Zalmoxidae, resulting in two new synonymies and 13 new combinations.
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PLoS ONE, Vol. 12, no. 11 (2017-11-30).

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RESEA RCH ARTICL E Light from dark: A relictual troglobite reveals a broader ancestral distribution for kimulid harvestmen (Opiliones: Laniatores: Kimulidae) in South America Abel Pe  rez-Gonza  lez 1 r, F. Sara Ceccarelli 1^a , Bruno G. O. Monte 2,3 , Daniel N. Proud 1^b , Ma  rcio Bernardino DaSilva 4 , Maria E. Bichuette 3 1 Divisio  n Aracno logõ  a, Museo Argentino de Ciencias Naturales "Bernard ino Rivadavia"Ð CONICE T, Buenos Aires, Argentina, 2 Programa de Po  s-Graduac Ë mo em Ecologia e Recursos Naturais, Universidad e Federal de Smo Carlos, Rodovia Washingto n Luis, Smo Carlos, Brasil, 3 Departamen to de Ecologi a e Biologia Evolutiva, Universidad e Federa l de Smo Carlos, Rodovia Washingto n Luis, Smo Carlos, Brasil, 4 Departamen to de Sistema  tica e Ecologia, Universidad e Federa l da Paraõ  ba, Paraõ  ba, Brasil ^a Current address: Departam ento de Biologõ  a de la Conservac io  n, CONACYT-C entro de Investigacio  n Cientõ  fica y de Educacio  n Superior de Ensena da, Carretera Ensena da-Tijuana, Ensena da B.C., Me  xico. ^b Current address: Moravian College, Environme ntal Studies & Sciences Program, Bethleh em, PA, United States of America. abelarac no@gmail.com Abstract A new troglobitic harvestman, Relictopiolus galadriel gen. nov et sp. nov., is described from Olhos d'A  gua cave, Itacarambi, Minas Gerais State, Brazil. Morphologic al characters, including male genitalia and exomorphol ogy, suggest that this species belongs to the family Kimulidae, and it appears to share the greatest similarities with Tegipiolus pachypus. Bayesian inference analyses of a molecular dataset strongly support the inclusion of this species in Kimulidae and confirm the hypothesized sister-group relationship between R. galadriel and T. pachypus. A time calibrated phylogeny indicates that these sister-taxa diverged from a common ancestor approximately 40 Mya, during the Paleogene. The current range of Kimulidae illustrates a remarkable disjunct distribution, and leads us to hypothesize that the ancestral distribution of Kimulidae was once much more widespread across eastern Brazil. This may be attributed to the Eocene radiation associated with the warming (and humidifying) events in the Cenozoic when the best conditions for evergreen tropical vegetation in South America were established and followed by the extinction of kimulid epigean populations together with the retraction of rain forests during the Oligocene to Miocene cooling. The discovery of this relictual troglobite indicates that the Olhos d'A  gua cave was a stable refugium for this ancient lineage of kimulids and acted as a "museum" of biodiversity. Our findings, considered collectively with the diverse troglofauna of the Olhos d'A  gua cave, highlight it as one of the most important hotspots of troglobite diversity and endemism in the Neotropics. Given the ecological stresses on this habitat, the cavernicolous fauna are at risk of extinction and we emphasize the urgent need for appropriate conservation actions. Finally, we propose the transfer of Acanthominua, Euminua, Euminuoides and Pseudominua from Kimulidae to Zalmoxidae, resulting in two new synonymies and 13 new combinations. PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 1 / 39 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 23(1 $&&(66 Citation: Pe  rez-Gonza  lez A, Ceccarelli FS, Monte BGO, Proud DN, DaSilva MB, Bichuette ME (2017) Light from dark: A relictual troglobite reveals a broader ancestral distribution for kimulid harvestm en (Opiliones : Laniatore s: Kimulidae) in South America. PLoS ONE 12(11): e0187919. https://do i.org/10.1371/j ournal.pone .0187919 Editor: William Oki Wong, Indiana Univers ity Bloomingt on, UNITED STATES Received: July 4, 2017 Accepted: October 27, 2017 Published: November 30, 2017 Copyright: ‹ 2017 Pe  rez-Gonza  lez et al. This is an open access article distributed under the terms of the Creative Commons Attributio n License, which permits unrestricte d use, distribu tion, and reproduction in any medium, provided the original author and source are credited. Data Availabilit y Statement: DNA sequences are deposited in GenBank (www.ncbi.nl m.nih.gov / genbank/); GenBank Accessio n Numbers for newly-gene rated sequences in Table 2. Funding: This study was funded by the grants PICT 2011-1007PI Martõ  n Ramõ  rez (APG); PICT 20150283-PI Martõ  n Ramõ  rez (APG) and PICT 20152202-PI Abel Pe  rez Gonza  lez (APG) from the Agencia Nacional de Promocio  n Cientõ  fica y Tecnolo  gica (ANPCyT) of Argentin a and the grants

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Introduction Morphological forms that are derived via adaptations to dark subterranean environments display a high degree of evolutionary convergence. Across diverse animal taxa, from fish and salamanders to arachnids and myriapods, troglomorphism is characterized by the loss of pigments and eyes, the elongation of appendages, and the elaboration of extra-optic sensory structures [1]. It is therefore no surprise that troglobitesÐspecies with life cycles exclusively in cavesÐ have instilled a sense of awe and bewilderment in taxonomists and evolutionary biologists for more than three centuries. Through the lens of a taxonomist, a troglobite represents an exquisite, seemingly bizarre taxon, incredibly divergent from even their closest epigean relatives, thus making them relatively easy to diagnose while simultaneously presenting a challenge to hypothesizing interspecific relationships. Meanwhile, through the lens of the evolutionary biologist, caves represent important laboratories of evolution [1,2] and cave faunas offer exceptional biological models to study the underlying evolutionary processes that drive adaptation and speciation to produce such remarkably convergent forms. Subterranean ecosystems have a unique combination of features including: i) truncated food webs, ii) relatively few lineages, iii) a high proportion of endemic species and many allopatric vicariant species, and iv) high level of relictual taxa [3]. The latter two features make troglobitic taxa ideal candidates for phylogenetic and biogeographic reconstructions. In the arachnid order Opiliones, known as harvestmen, there is a considerable number of relictual troglobites, however, most species are poorly studied and others remain undiscovered. For example, Rambla and Juberthie [4] recognized a total of 82 troglobitic harvestmen worldwide, but that number is outdated and grossly underestimated. Harvestmen are common inhabitants of subterranean ecosystems and are well represented in all classification categories of subterranean fauna (sensu Trajano and Carvalho [5]). Troglobite harvestmen species could play an important role in understanding the complete phylogenetic history of their respective lineages. For example, the highly troglomorphic species n (Goodnight & Goodnight, 1977) from Belize was originally described in the Neotropical family Stygnommatidae, but new evidence, based on studies of genitalic morphology and molecular phylogenetic analyses, revealed that it was the first American representative of the Pyramidopidae, which was previously thought to be endemic to Africa [6]. The case of n clearly illustrates the challenges of interpreting systematic relationships when faced with highly convergent morphologies, and it emphasizes the need for an integrative approach (.., fine morphology, molecular phylogeny) to investigate the evolutionary history of troglobitic species. Until the present study, the Brazilian troglobitic opiliofauna consisted of nine described species, all Laniatores±eight species of Gonyleptidae (rnn Kury, 2008; n Kury, 2008; n Pe  rez-Gonza  lez and Kury, 2002; n Hara and Pinto-da-Rocha, 2008; Pinto-da-Rocha, Fonseca-Ferreira and Bichuette, 2015; Pinto-da-Rocha, 1997; n S Ï ilhavy  , 1974; Kury and Pe  rez-Gonza  lez, 2008) and one species of Escadabiidae ( H. Soares, 1966). A number of other troglobitic harvestmen species are known, but are awaiting formal taxonomic description [7±9]. One species of Cyphophthalmi, DaSilva, Pinto-da-Rocha and Giribet, 2010, was collected exclusively from caves in the Brazilian state of Para  , but due to the absence of troglomorphic features, it was not considered a troglobite. The occurrence of this species in caves was attributed to the need for shelter from the dry climatic conditions in this region and it is assumed that the species also inhabits adjacent forests [10]. No troglobitic Eupnoi from Brazilian caves are known to date. During surveys and studies of the Brazilian cave fauna, conducted by the "Laborato  rio de Estudos Subterra à neos" (Laboratory of Subterranean Studies) of the Federal University of So A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 2 / 39 PIP 1122015010 0672CO-PI Andre  s OjangurenAffilastro (APG) and PUE 2017-2021PI Martõ  n Ramõ  rez (APG) from the Consejo Nacional de Investigac iones Cientõ  ficas y Te  cnicas (CONICET) , Argentina. This work was also partially supporte d by postdoctoral scholarship grants (resolution 4817/2013 ) from Consejo Nacional de Investigac iones Cientõ  ficas y Te  cnicas (CONICET) , Argentina (FSC and DNP) and the scholarship PQ 303715/20 11-1 from the Conselho Nacional de Desenvolvi mento Cientõ  fico e Tecnolo  gico (CNPq), Brazil (M.E.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests : The authors have declared that no competing interests exist.

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Carlos, Brazil, a tiny depigmented harvestmen species was collected from the Olhos d' A  gua cave, a biospeleologically iconic Brazilian cave in Peruac Ëu, Itacarambi, Minas Gerais State. The specimens belong to a new genus and a new species of the family Kimulidae, herein formally described. A molecular phylogenetic analysis was used to test taxonomic hypotheses and provide a basis for a discussion of the biogeographic history of Kimulidae. Materials and methods Sampling locality Olhos d'A  gua cave [geographic coordinates 15Ê 6'49.32"S, 44Ê10'10.56"W] is located in Peruac Ëu Caves National Park (PCNP), northern Minas Gerais State, Itacarambi municipality (Fig 1). The karst region of PCNP is composed of outcrops with a predominance of carbonate karst areas developed on Proterozoic rocks of the Bambuõ  Group, mainly limestone and dolomites (Janua  ria/Itacarambi Formation sensu Pilo  and Kohler [11]). Within the boundaries of the PCNP, the Peruac Ëu river runs through a valley with large walls, pipes and sinkholes forming its canyon. Peruac Ëu River Basin is a left tributary of So Francisco River (Pilo  and Kohler 1991). The PCNP is situated in the transition between the Cerrado and Caatinga morphoclimatic domains [12] and, according to the Koppen-Geiger classification [13], the climate is tropical semiarid, with a well-defined dry period between April and September, an average annual temperature of 24ÊC and average annual rainfall of 800 mm [14]. Olhos d'A  gua cave has a horizontal projection of approximately 9,100 m and consists of a long and sinuous conduit. The dimensions of galleries vary from low passages to large rooms, with few upper conduits. Although the area has the lowest amount of annual rainfall in the region, the drainage in the cave is perennial. On the other hand, intense flooding may occur in the cave during the rainy season. The main cave entrance is a resurgence, which is not located within the PCNP limits. The substrate and available microhabitats for the terrestrial fauna are predominantly formed by sand, boulders and other rocky substrates, silt, and with occasional vegetable debris deposited along the river banks (Fig 2). Specimen collection and repositories The field study and collection of specimens at the Peruac Ëu Caves National Park (PCNP) were carried out under the SISBIO permit number 28992±3 (to MEB) issued by the Instituto Chico Fig 1. Locality of the Olhos d'A  gua cave in South America. (A), (B) Photograph s of surrounding vegetation . PCNP: Peruac Ë u Caves National Park. https://do i.org/10.1371/j ournal.pone .0187919.g00 1 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 3 / 39

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Mendes de Conservac Ëo da Biodiversidade (ICMBio) of the Ministe  rio do Meio Ambiente (MMA), Brazil. The field study did not involve officially endangered or protected species. Specimens were collected by active search and were preserved in 80% and 96% ethanol for morphological study and DNA extraction, respectively. Types are deposited in the Laborato  rio de Estudos Subterra à neos, Universidade Federal de So Carlos, So Carlos, Brazil (LES/UFSCAR), Museu de Zoologia, Universidade de So Paulo, So Paulo, Brazil (MZUSP) and Divisio  n de Aracnologõ  a in the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia", Buenos Aires, Argentina (MACN-Ar). Other specimens used in this study are deposited at: Colec Ëo de Histo  ria Natura da Universidade Federal do Piauõ  (CHNUFPI), Universidade Federal da Paraõ  ba (UFPB), Museu Nacional/Universida de Federal do Rio de Janeiro (MNRJ), Instituto Butantan, So Paulo (IBSP), National Museum of Natural History, Smithsonian Institution (NMNH), Zoologisk Museum Universita È t Kbenhavn (ZMUC), Naturmuseum Senckenberg Sektion Arachnologie (SMF). Maps All maps were generated using the free, open source geographic information system software QGIS 2.16.3 (http://www.qgis.org/); the shape files are freely available from the Ministe  rio do Meio Ambiente (MMA) web page (http://www.mma.go v.br/governanca-ambiental/ geoprocessamento). Nomenclatural acts This article conforms to the requirements of the amended International Code of Zoological Nomenclature. All nomenclatural acts contained within this published work have been registered in ZooBank. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the Fig 2. Cartographi c mapr of the Olhos d'A  gua cave. (I), (II), (III) Photograph s corresponding to cave cross sections indicated on the detailed cartogr aphic map. (A), (B) Sites in the cave where Relictopiolu s galadriel gen. nov., sp. nov. was captured . Courtesy of the Bambuõ  Speleologi cal Group. https://do i.org/10.1371/j ournal.pone .0187919.g00 2 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 4 / 39

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associated information viewed by appending the LSID to the prefix ªhttp://zoobank.org/º. The LSID for this publication is: urn:lsid:zoobank.org:pub:2ACD230 C-33E9-4461-A40D7FE418F94B21. Specimen preparations Ethanol preserved specimens were photographed with a Leica DFC 290 digital camera attached to a Leica M165C stereomicroscope (at MACN), and different focal planes were combined using Helicon Focus Pro (www.heliconsoft.com). The picture of the holotype of n (SMF 9906896) was taken with a Sony DSC-V1 digital camera and no focal planes were combined. Color descriptions follow Kury and Orrico [15]. Male genitalia preparation follows Acosta et al. [16], with temporary mounts embedded in glycerol. Line drawings of male genitalia were made using a camera lucida attached to an Olympus BH-2 compound microscope (at MACN) and were digitized using Corel Draw X7. Figures were edited using Photoshop CS5 or Corel Draw X7. For SEM, dissected body parts were dried using Critical Point Drying and mounted on adhesive copper tape (EMS 77802; Electron Microscopy Sciences) affixed to an aluminum stub. Uncoated SEM preparations were examined using a FEI Quanta 250 (at the UFSCar). Taxon sample Using the dataset from Cruz-Lo  pez et al. [6] (88 terminals), we added new sequence data for 24 terminal taxa representing Escadabiidae (10 new terminals), Kimulidae (6 new terminals), Samoidae (3 new terminals), and Zalmoxidae (5 new terminals). The dataset was particularly strengthened by the addition of Escadabiidae and Kimulidae representatives and included more terminals of these Zalmoxoidea than all the previous molecular phylogenetic analyses of Laniatores. Specimen data for these 24 taxa included the molecular phylogenetic analysis, including voucher numbers and locality data, are listed in Table 1. Information for the rest of the terminals can be found in Sharma and Giribet [17] and Cruz-Lo  pez et al. [6]. DNA sequencing and alignment DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit, digesting tissue from one or two legs at 56Ê C over-night with Proteinase K and following the manufacturer's protocol. The DNA was then used to amplify and sequence four molecular markers, namely fragments belonging to the mitochondrial cytochrome c oxidase subunit I (COI) gene, and the nuclear histone H3 (H3), 18S rRNA (18S) and 28S rRNA (28S) genes. Polymerase Chain Reactions (PCR) were carried out to amplify the four gene regions, setting up a master mix containing 1.5l x10 PCR Buffer (Thermo Scientific), 10 moles MgCl 2 , 0.25 moles of each dNTP, 0.4 moles of each primer, 0.1 l Taq Polymerase (Thermo Scientific), 0.5 l BSA, 1±2 l genomic DNA and ddH 2 O to bring the final volume to 15 l. The primers used for amplification included LCO1490-HCOout out (COI), H3aF-H3aR (H3), 1F-5R (18S), and 28Srd4.8a28Srd7b1 (28S) following Sharma and Giribet [17]. Thermal cycling included an initial denaturing step at 95Ê C for 3 minutes, followed by 15 cycles of 30 seconds at 95Ê C, 30 seconds at the annealing temperature (51Ê C for nuclear and 45Ê C for mitochondrial gene fragments) and 45 seconds at 72Ê C; an additional 20 cycles were run with the annealing temperature lowered by 3Ê C and a final extension step of 10 minutes at 72ÊC was executed. PCR products were purified using ExosAP (Thermo Scientific) following the manufacturer's protocol and sent for sequencing to Macrogen Inc., Korea. The chromatograms of the sequences were edited in Sequencher v. 4.1.4 (GeneCodes corp.), where the protein-coding gene fragments COI and H3 were checked for stop-codons. A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 5 / 39

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Table 1. Specimen s included in this study for which new sequence data were obtained , including voucher numbers and collection details. Family Genus specie s Reposito ry DNA voucher Name on the tree Collection locality details Escadabiid ae Gen. ind. sp. ind. MACN-Ar Es01 Escadabiid ae_Es01 ARGENTIN A, Misiones Province, Parque Provincial Urugua -õ  , 25Ê 48'32.94"S , 54Ê 1'0.70" W, 377 masl, 11.iii.2015 , A. Pe  rez Gonza  lez, Daniel N. Proud, Sara Ceccarell i and A. MartinezAquino. Escadabiid ae Gen. ind. sp. ind. MACN-Ar Es02 Escadabiid ae_Es02 ARGENTIN A, Misiones Province, Parque Provincial Urugua -õ  , 25Ê 48'32.94"S , 54Ê 1'0.70" W, 377 masl, 11.iii.2015 , A. Pe  rez Gonza  lez, Daniel N. Proud, Sara Ceccarell i and A. MartinezAquino. Escadabiid ae Gen. ind. sp. ind. MACN-Ar Es03 Escadabiid ae_Es03 ARGENTIN A, Misiones Province, San Ignacio, Reserva Privada Osununu  , Janua ry 2015, C.I. Paris, P.E. Hanish and A.F. Sanchez Restrepo . Escadabiid ae Brotasus cf. megalobunus MACN-Ar Es04 Brotasus_c f_megalob unus_Es04 BOLIVIA: Santa Cruz: Prov. Guarayos : Concesio  n Forestal La Chonta; S 15Ê 42' 42", W 62Ê 46' 20" 330 m. 26±30 Oct. 2010. Col. C. Grismad o, M. R. Vacaflores & M. Pe  rez. Escadabiid ae Brotasus sp. ind. LES Es17 Brotasus_s p_Es17 BRAZIL, Mato Grosso State, Distrito da Cerqui nha, munõ  cipio de Nobres, Duto do Quebo  , 14Ê26'45 .7"S, 56Ê 01'15.9" W, 330 masl, 23.ix.2015 , M. E. Bichuette , A. Chagas-Jr , D.M. von Schimons ky. Epõ  geo. (field code MT 397). Escadabiid ae Baculiger us? sp. ind. CHNUFPI 1094 Es06 Baculiger us?_sp_Es 06 BRAZIL, Piauõ  , Castelo do Piauõ  , Fazenda Bonito, Espanh a-ChileBrasil Rochas Ornamen tais do Brasil LTDA, S 5Ê 13'33.4", W 41Ê41'46.8", 10.v.2013 , L.S. Carvalho et al. Escadabiid ae Gen. ind. sp. ind. UFPBÐ OP0035 Es07 Escadabiid ae_Es07 BRAZIL, Alagoas State, Mata do Brejo, Usina Serra Grande, Smo Jose  do Laje, 8Ê58'48. 00"S, 35Ê 51'0.00" W, 16.ii.2012 . A.M. DeSouza et al. Escadabiid ae cf. Spaeleole ptes sp. ind. LES Es08 cf_Spaelol eptes_Es08 BRAZIL, Bahia State, Ourola à ndia, Gruta da Fazenda Caldeirmo, (11Ê 00'56"S, 40Ê39'58" W), 772 masl, 17. vi.2015, J. E. Gallmo. (field code GR055). Escadabiid ae Escadabius ventricalcarat us UFPB AL-12 Es11 Escadabiu s_ventricalc aratus_Es11 BRAZIL, Pernambu co State, Reserva Particular do Patrimo à nio Natural Frei Caneca, 8Ê40'48.0 0"S, 35Ê51'0. 00" W, 5-7.viii.201 1, M.B. DaSilva et al. Leg. Escadabiid ae Baculiger us cf. littoris MNRJ 08841 Es14 Baculiger us_cf_littori s_Es14 BRAZIL, Bahia State, Corac Ë mo de Maria [12Ê15' 17.55"S 38Ê 48'29.33"W , 300 masl], Oct.2015, A. Kury & O. Villarreal Manzanilla . Kimulidae Tegipiolus pachypus UFPB AL-14 Es10 Tegipiolus _pachypus _Es10 BRAZIL, Pernambu co State, Caruaru , Parque Natural Municipal Professor Jomo Vasconce los Sobrinho (Brejo dos Cavalos), 8Ê 21'36.00"S , 36Ê 1'12.00 "W, 830 masl, x.2001, A.M. Souza & A. Lira. (Continue d ) A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 6 / 39

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Table 1. (Continue d ) Family Genus specie s Reposito ry DNA voucher Name on the tree Collection locality details Kimulidae Relictopiolus gen. nov. galadriel sp. nov. LES Es18 Relictop iolus_galadri el_Es18 BRAZIL, Minas Gerais State, Itacaramb i, Peruac Ë u Caves National Park, Olhos d' A  gua cave, 15Ê 6'49.32" S, 44Ê10'10. 56"W, 24. x.2015, Monte, B.G.O., Zepon, T. Kimulidae Relictopiolus gen. nov. galadriel sp. nov. LES Es19 Relictop iolus_galadri el_Es19 BRAZIL, Minas Gerais State, Itacaramb i, Peruac Ë u Caves National Park, Olhos d' A  gua cave, 15Ê 6'49.32" S, 44Ê10'10. 56"W, 24. x.2015, Monte, B.G.O., Zepon, T. Kimulidae Kimula sp. ind. NMNH Es21 Kimula_ sp_Es21 CUBA, Guanta  namo Province, Baracoa, Alejandr o de Humboldt National Park, El Yunque, 20Ê 20'42.04"N , 74Ê33'59. 11"W, 370 masl, 5.iv.2012 , CarBio Team. (CarBio field code CU-15) Kimulidae Kimula sp. ind. NMNH Es22 Kimula_ sp_Es22 PUERTO RICO, Cambalac he, Bosque Cambalac he, 3.iii.2012 , CarBio Team. (CarBio field code PR200). Kimulidae Gen. ind. sp. ind. MACN-Ar Es24 Kimulidae_E s24 PANAMA : Cocle  , Parque Nacional General de Divisio  n Omar Torrijos Herrera, El Cope, 1 hectare PANCODI NG inventory [N 08Ê 40'5.1", W 80Ê35'33 .3"], 760 masl, 49.vi.2008 , M. Arnedo, L. Benavid es, G. Hormiga, F. Labarque, M. Ramõ  rez Col. (field code STC1N6R) Zalmoxida e Gen. ind. sp. ind. MNRJ 08843 Es13 Zalmoxid ae_Es13 BRAZIL, Bahia State, Vila Bela da Santõ  ssima Trindade , Fazenda Barraco Alto [14Ê56' 18.64"S, 60Ê 0'33.84" W, 222 masl], 14.xi.201 5, A. Chagas-J r. & A. Kury. Zalmoxida e Gen. ind. sp. ind. MNRJ 08842 Es15 Zalmoxid ae_Es15 BRAZIL, Mato Grosso State, Porto Estrela, Estac Ë mo Ecolo  gica da Serra das Araras [15Ê30'3 .26"S, 57Ê 9'57.35" W, 200 masl], 16.xi.201 5, A. Chagas-J r. & A. Kury. Zalmoxida e Gen. ind. sp. ind. LES Es16 Zalmoxid ae_Es16 BRAZIL, Mato Grosso State, Distrito da Cerqui nha, munõ  cipio de Nobres, Toca do Quati, 14Ê27'11 .2"S, 55Ê 59'12.6" W, 392 masl, 22.ix.2015 , M. E. Bichuette , A. Chagas-Jr , D.M. von Schimons ky. Zona de entrada / declive-fo lhic Ë o (field code MT 143). Zalmoxida e Gen. ind. sp. ind. NMNH Es23 Zalmoxid ae_Es23 PUERTO RICO, Villalba, Toro Negro, 18Ê10'22. 72"N, 66Ê 29'30.47"W , 889 masl, 27-29. vii.2011, CarBio Team (CarBio field code PR 17 CR) Zalmoxoid ea Family and Gen. ind. sp. ind. NMNH Op019 Zalmoxoi dea gen sp. Op019 PUERTO RICO, Rio Grande, El Yunque, El Verde, 18Ê19'18 .08"N, 65Ê49'11. 67"W, 323 masl, 16-19. vii.2011, CarBio Team (CarBio field code PR001) (Continue d ) A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 7 / 39

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The edited sequences were combined with sequences from previous studies [17,18] for a total of 124 ingroup and outgroup taxa. Alignments were constructed in the MAFFT v. 7 [19] web service, using the ªAutoº strategy and a gap opening penalty of 1.53. For the protein-coding genes, COI and H3, their translation to amino acids was checked in MEGA 7.0.14 [20] and the hypervariable regions of 28S which could not be aligned with confidence were removed for the phylogenetic analyses using the ªstringentº settings of the Gblocks server online [21]. Newly-generated sequences were submitted to GenBank and their accession numbers can be found in Table 2. Phylogenetic analysis and divergence time estimation Phylograms and chronograms for our focal taxa and selected outgroups were obtained through Bayesian inference (BI). As site-specific nucleotide saturation can have a negative influence on phylogenetic inference, the nucleotide composition homogeneity was tested for each data matrix, including matrices of individual codon positions for the protein coding gene fragments. The program TreePuzzle [22] was used to evaluate nucleotide compositional homogeneity using a chi-squared metric. This test indicated that the third codon positions of both COI and H3 were highly saturated and were therefore excluded. Furthermore, the nucleotide substitution model and partitioning strategy for the combined (concatenated) data matrices was evaluated in PartitionFinder v. 1.1.1. [23] (for details see Table 3). A phylogram of the concatenated, partitioned data was obtained using Markov Chain Monte Carlo (MCMC) simulations in MrBayes v. 3.2.6 [28], with the corresponding substitution model set for each partition and unlinking all parameters. Two independent MCMC runs with four chains each were executed for 50 million generation, sampling every 5000 generations and, after checking for correct mixing of the chains and large enough effective sample sizes (>200), a consensus tree was built using a 10% burn-in, and nodal support was assessed through posterior probabilities (PP). The time-calibrated tree was built in BEAST v. 1.8.3 [29] using the same partitioning strategy and nucleotide substitution models as in MrBayes. A birth-death prior was set for the trees, linked across the partitions to obtain a single topology. Clock priors were unlinked per marker and set as uncorrelated relaxed clocks with lognormal distributions and estimated rates. Node age estimates were based on a combination of fossil evidence and secondary Table 1. (Continue d ) Family Genus specie s Reposito ry DNA voucher Name on the tree Collection locality details Samoida e Hummelin ckiolus sp. ind. NMNH Op020 Hummeli nckiolus_sp_ Op020 CUBA, Santiag o de Cuba Province, Siboney , 19Ê57'3 8.88"N, 75Ê 42'27.36"W , 30 masl, 1.iv.2012, CarBio Team (CarBio field code CU007) Samoida e Hummelin ckiolus sp. ind. NMNH Op7048 Hummeli nckiolus_sp_ Op7048 DOMINI CAN REPUB LIC, La Vega Province, Los Tablones, 19Ê 03'18.58"N , 70Ê53'19. 18"W, 1304 masl, 28.vi.201 2, CarBio Team (CarBio field code DR030) Samoida e Hummelin ckiolus sp. ind. NMNH Op7050 Hummeli nckiolus_sp_ Op7050 DOMINI CAN REPUB LIC, La Vega Province, Valle Nuevo, 18Ê 50'46.79"N , 70Ê44'26. 30"W, 2982 masl, 26.vi.201 2, CarBio Team (CarBio field code DR022) https://do i.org/10.1371/j ournal.pone .0187919.t001 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 8 / 39

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calibrations. Three calibrations based on the estimated ages of fossils belonging to specific groups were set as minimum node age constraints. The first calibration was based on an available fossil, n Cokendolpher and Poinar, 1998 [30], setting a minimum age of 16 Million years (Ma) for the most recent common ancestor (mrca) of the three n species in our dataset. A minimum age of 16 Ma was also set for the mrca of the three species in this study, based on the fossil ? sp. [31]. Finally, based on a fossil of Selden et al., 2016 [32], belonging to the family Epedanidae, a minimum age of 99 Ma was set for the mrca of the infraorder Grassatores minus the superfamily Phalangodoidea, given the sparse sampling of epedanids and the lack of resolution between said family and its sister groups in our phylogeny (see Results). The secondary calibration involved setting the minimum age of the zalmoxid taxa in this study to 87.3 Ma, based on the Table 3. Partitioning strategy and nucleotide substitutio n models selected for each partition by PartitionFind er for Bayesian phylogenet ic analyses . Partition Substitutio n model Referen ce COI codon position 1 GTR+I+G [24] COI codon position 2 GTR+I+G [24] H3 codon position 1 GTR+I+G [24] H3 codon position 2 K81 [25] 18S rDNA TIMef+I+G [26] 28S rDNA TrNef+I+ G [27] https://d oi.org/10.1371/j ournal.pon e.0187919.t00 3 Table 2. GenBank accession numbers for newly-gener ated sequence s. Taxon DNA voucher COI H3 18S 28S Escadabiid ae: gen. ind. sp. ind. Es01 MF687049 MF687066 MF687 005 MF687026 Escadabiid ae: gen. ind. sp. ind. Es02 MF687050 ÐÐ MF687 006 MF687027 Escadabiid ae: gen. ind. sp. ind. Es03 MF687051 ÐÐ ÐÐ MF687028 Escadabiid ae: Brotasu s cf. megalo bunus Es04 ÐÐ MF687067 MF687 007 MF687029 Escadabiid ae: Brotasu s sp. Es17 ÐÐ MF687068 MF687 008 MF687030 Escadabiid ae: Baculig erus? sp. Es06 MF687052 MF687069 MF687 009 MF687031 Escadabiid ae: Gen. ind. sp. ind. Es07 ÐÐ ÐÐ MF687 010 ÐÐ Escadabiid ae: cf. Spaeleo leptes sp. Es08 MF687053 ÐÐ ÐÐ MF687032 Escadabiid ae: Escadabi us ventricalcar atus Es11 ÐÐ MF687070 MF687 011 MF687033 Escadabiid ae: Baculig erus cf. littoris Es14 ÐÐ MF687071 MF687 012 MF687034 Kimulidae: Tegipiolus pachypus Es10 MF687054 ÐÐ MF687 013 MF687035 Kimulidae: Relictop iolus galadriel gen. nov. et sp. nov. Es18 MF687055 ÐÐ MF687 014 MF687036 Kimulidae: Relictop iolus galadriel gen. nov. et sp. nov. Es19 MF687056 ÐÐ MF687 015 MF687037 Kimulidae: Kimula sp. Es21 MF687057 MF687072 MF687 016 MF687038 Kimulidae: Kimula sp. Es22 MF687058 MF687073 MF687 017 MF687039 Kimulidae: gen. ind. sp. ind. Es24 ÐÐ ÐÐ ÐÐ MF687040 Zalmoxida e: gen. ind. sp. ind. Es13 MF687059 MF687074 MF687 018 MF687041 Zalmoxida e: gen. ind. sp. ind. Es15 MF687060 ÐÐ MF687 019 MF687042 Zalmoxida e: gen. ind. sp. ind. Es16 ÐÐ MF687075 MF687 020 MF687043 Zalmoxida e: gen. ind. sp. ind. Es23 MF687061 MF687076 MF687 021 MF687044 Zalmoxoid ea gen. sp. Op019 MF687062 ÐÐ MF687 022 MF687045 Samoida e Humme linckiolus sp. Op020 MF687063 ÐÐ MF687 023 MF687046 Samoida e Humme linckiolus sp. Op7048 MF687064 ÐÐ MF687 024 MF687047 Samoida e Humme linckiolus sp. Op7050 MF687065 ÐÐ MF687 025 MF687048 https://do i.org/10.1371/j ournal.pone .0187919.t002 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 9 / 39

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minimum range for the 95% highest posterior density (HPD) of the age obtained for this family by Sharma and Giribet [18]. With these priors, two independent MCMC runs were executed for 100 million generations each, sampling every 10,000 generations and combining the sampled trees from the two runs using LogCombiner (part of the BEAST package). After verifying that the ESS of all parameters was >200 in Tracer v. 1.5 [33], TreeAnnotator (part of the BEAST package) was used to choose the maximum clade credibility tree, with node information (posterior probabilities, 95% HPD of node ages etc.) based on all sampled trees minus 10% burn-in. Phylogenetic analyses both in MrBayes and BEAST were repeated including the third codon positions of COI and H3 to corroborate the effect of their initial exclusion on topology, nodal support and node age estimates. Results and discussion Phylogeny The information for the DNA sequence data matrices used for the phylogenetic inferences in this study can be found in Table 4. The phylogenetic topologies obtained by Bayesian Inference implemented in MrBayes (phylogram; Fig 3, S1 Fig) and BEAST (chronogram; Fig 4, S2 Fig) were similar, although certain nodes differed in whether the monophyly of the group was supported by posterior probabilities, or not. Similarly, there were no substantial topological or node-age differences when the saturated (COI and H3 third codon positions) data was included (S3 and S4 Figs), except that with the latter datasets, the overall nodal support values were lower. We therefore restrict the presentation and discussion of results to the phylogenetic trees obtained with the datasets excluding third codon positions. Discussions of the higher-level systematic of Laniatores are beyond the scope of the present contribution, but some findings do warrant a brief mention. Our phylogram (Fig 3) indicates strong support for the monophyly of all non-phalangodid Grassatores (PP = 1.0) corroborating previous findings that have utilized this dataset (.. [6,17,34]). One remarkable congruence between our phylogram and the Opiliones transcriptomic-based phylogenetic hypothesis of Ferna  ndez et al. [35] is the the sister group relation of Assamioidea with the Samooidea+Zalmoxoidea clade (albeit with low support, PP = 0.77). The chronogram failed to recover this relationship, placing Assamiidae as sister to Pyramidopidae, but with no support (PP = 0.2). Also consistent with previous studies [6,17,35,36], we recovered the clade Samooidea+Zalmoxoidea with high support (phylogram with PP = 0.96 and chronogram with PP = 1) despite the unstable internal relationships of this clade (Figs 3 and 4). To achieve the objective of our study, and test our hypothesis regarding the familial placement of the new troglobitic species, we generated a dataset to include the broadest taxon sampling of the species diversity for Kimulidae and Escadabiidae to date, adding 16 new terminals. Table 4. Nucleotid e composit ion of the four DNA data matrices of the markers used for phylogen etic inferences in this study, showing the number of taxa (ntax), the total number of sites in the alignment (nsites) and the number of variable (nvar) and parsimo ny-informa tive (npis) sites. Marker ntax Nsites Nvar npis COI 85 816 548 494 H3 87 327 142 136 18S 122 1769 257 257 28S 123 2174 498 325 https://d oi.org/10.1371/j ournal.pon e.0187919.t00 4 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 10 / 39

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Fig 3. Phylogram of Grassa tores obtained by Bayesian inference analysis, conducte d in MrBayes , using the complete concatena ted dataset. Support values at nodes represent posterior probabilitie s (PP). Nodes with PP0.50 are collapsed . Colors represent the current superfami lial divisions (sensu Fernandez et al. [35]). The monophyle tic family Kimulidae is enclosed in a box. https://doi.o rg/10.1371/j ournal.pone .0187919.g003 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 11 / 39

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We obtained strong support for the monophyly of Kimulidae based on the MrBayes analysis but not for the BEAST analysis (PP MRBAYES = 0.99; PP BEAST = 0.56). Nested within the monophyletic Kimulidae was our focal taxon, the troglobite !n gen. nov., sp. nov., recovered as the sister-taxon to n Roewer, 1949, with strong support (PP MRBAYES = 1.0; PP BEAST = 0.99). The age estimate for the most recent common ancestor of Fig 4. Chronogr am (partim, Zalmoxoidea ) obtained by Bayesian inference analysis, conducte d in BEAST, using the complete concate nated dataset. (A) Chronogram with support values at nodes represent posterior probabilitie s and blue bars repres ent the 95% Highest Posteri or Densit ies (HPD) around the estimated age of the most recent common ancesto r. Colored branches and names of taxa repres ent the family Kimulidae. Below the chronogram , a geologic time scale of the Cenozoic Era (present-65 Ma) is shown. The vertical red-dotted line indicates the divergenc e time (40.1 Ma) between Relictopiolu s galadriel gen. nov., sp. nov. and Tegipiolus pachypus Roewer, 1949, projecte d over the geologic time scale with a blue shaded box that indicates the 95% HPD. https://do i.org/10.1371/j ournal.pone .0187919.g00 4 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 12 / 39

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these two species is 40.12 million years ago (Ma; 95% HPD: 19.06±64.27) (Fig 4) suggesting that they diverged sometime during the Paleogene. Despite our efforts to greatly increase the taxon sampling within these zalmoxoid lineages, the sister-group of Kimulidae remains unclear, possibly obscured by the poorly understood diversity of the Escadabiidae which were not recovered as monophyletic in either analysis. However, the maximum clade credibility tree from BEAST places Kimulidae as sister to a monophyletic Escadabiidae, albeit without valid nodal support (Fig 4). Careful examination of morphological characters (e.g., genitalia) and a denser taxon sampling will be necessary to study the deeper phylogenetic relationships within these families, and their relationships within the Samooidea+Zalmoxoidea clade. Disjunct distributions The Olhos d'A  gua cave, the type-locality of !n gen. nov., sp. nov., is situated along the transition zone between the Caatinga and Cerrado ecoregions and is surrounded by a shrubby and dry vegetation (Figs 1A, 1B and 5). However, the vegetation classification system indicates a deciduous forest [37] covering the limestone lithology of the region and following the So Francisco river, which is considered part of the Atlantic Forest biome. Historical biogeography of biota living in this region indicates a relationship between the area of endemism of the eastern Atlantic forest of Bahia [38,39] and the south-eastern Amazon forest, as these two large blocks of Neotropical rain forests were once connected [38]. Approximately 1000 km to the northeast of the Olhos d'A  gua cave, in the northeastern regions of the Brazilian Atlantic Rain Forest biome, . n inhabits coastal humid forests and inland semidecidous forests (Fig 5). The coastal plain and the eastern slopes of the plateau (Serra da Borborema) are covered by Ombrophilous Forest, characterized by a wet climate, while the interior and northern regions have semideciduous forests that experience a marked dry season. Records indicate that the species can be found in semideciduous enclaves of mesic forest at higher altitudes (> 600 m), known as "Brejos de Altitude", but not in the surrounding lowland habitats of dry Caatinga scrubland. The range of . n coincides with the ranges of many animals and plants that are endemic to the Pernambuco interior and coastal forests [40] and thus appears to be related to the Bahia area of endemism [41,42]. While the 1000 km expanse that separates !n and (Fig 5) is quite astonishing, what is more remarkable is the geographic disjunction between these sister-taxa and all other Kimulidae, which exhibit peak diversity in northwestern South America and the West Indies (Fig 6). Specifically, the kimulid genera " Roewer, 1949 and #n Gonza  lezSponga, 1998, occur in Venezuela, mainly in humid and high-altitude habitats, and Goodnight and Goodnight, 1942 and " Avram, 1973, occur in Antillean islands (Cuba, Puerto Rico, Hispaniola and Virgin Islands). A representative of Kimulidae (undetermined genus and species) is herein recorded for Panama and other unpublished records denote the presence of this family in Chiapas, Mexico (representing the northern limit of the family in the continental Americas), Trinidad and Tobago (MCZ Collections Database, http://mczbase. mcz.harvard.edu/) Colombia and Brazil (Manaus and CoariÐ360 Km W of ManausÐAmazonas State, Põ  o Colmenares pers. comm; Caracaraõ  , Roraima State, MCZ Collections Database, http://mczbase.mcz.harvard .edu/) (Fig 6). Thus, geographically, the nearest kimulid species to !n and is located more than 2,000 km to the northwest in Manaus, Brazil. Biogeography of Kimulidae The phylogenetic relationships of Kimulidae appear to reflect the general biogeographic hypotheses of the major Neotropical areas defined by Morrone [43]. We recovered a clade A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 13 / 39

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consisting of and " from the Antilles and " from Venezuela, illustrating a shared evolutionary history between the Antillean subregion and Pacific dominion of sensu Morrone [44]. An undetermined genus from Panama  , which undoubtedly belongs to Kimulidae, was not recovered as the sister group to the Venezuelan taxa (Fig 4). This suggests that there is a complex evolutionary history for Kimulidae in the Andean region, but broader taxon sampling is required. The distributional ranges of !n and fall within the Parana  dominion of Morrone's classification [44], which is composed of Atlantic Forest biota. Fauna in the Atlantic Forest is related to that of the South-eastern Amazonia and Chacoan dominions, represented by the drier Caatinga and Cerrado biomes [44,45]. Thus, the two major clades of Kimulidae inhabit biogeographic regions that are not directly related in the context of evolutionary and biogeographic history in South America [43]. There are at least two plausible, nonexclusive explanations for the biogeographic patterns observed for !n and : 1) that we have a poor understanding of the diversity of Kimulidae and its species distributions, and 2) that these two species represent relicts of a once widespread ancestral distribution that has undergone range reduction due to extinction. First, there is undoubtedly a large gap in our taxonomic knowledge of this group. There may Fig 5. Current distribution of Relictopiol us galadriel gen. nov., sp. nov. (triangle) and Tegipio lus pachypus Roewer, 1949 (dots). The vegetation types (sensu the vegetationa l classif ication system proposed by the Instituto Brasileiro de Geografia e Estatõ  stica [37]) are delimited by color. https://do i.org/10.1371/j ournal.pone .0187919.g00 5 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 14 / 39

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be myriad species inhabiting tropical forests in the southern and eastern parts of the Amazon Rainforest that remain undiscovered and undescribed. However, in the Amazon Forest near Manaus, after conducting multi-year intensive surveys within a large reserve, Kimulidae appear to be extremely rareÐonly a single species was discovered, based on a single specimen (Põ  o Colmenares pers. comm.). Additionally, of the 71 species recorded for the Brazilian state of Amazonas and 45 species recorded for the state of Para  , there are no known species of Kimulidae [46]. Therefore, although the list of known species is by no means exhaustive in these two states, the paucity of records for even a single species for Kimulidae leads us to hypothesize that these two taxa represent a case of relictualism. Biogeography of Relictopiolus + Tegipiolus The split between !n and also appears to result from relictualism. The 1000 km expanse that separates these species is occupied by the Caatinga bioregion, a semiarid barrier (Fig 5). Based on natural history observations and available records, species of Kimulidae appear to primarily occupy and thrive in moist environments. Thus, the occurrence of !n may be a relict of a widespread ancestral distribution throughout tropical forests that stretched from the Amazon to the Bahia Atlantic Forest±covering what are now the semiarid habitats of the Caatinga with tropical habitats. As with most harvestmen taxa that exhibit peak diversity in tropical regions, the Kimulidae are poorly studied and the diversity of the family is extremely underestimated. There are many species that remain to be described, and only then can we begin to formulate and test stronger hypotheses regarding the systematics and biogeography of this family and its interfamilial relationships within Zalmoxoidea. Fig 6. Known distributio n of the family Kimulida e detailing the geogra phic range of each genus. https://do i.org/10.1371/j ournal.pone .0187919.g00 6 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 15 / 39

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The geographic distance between and !n is correlated with a deep genetic divergence that is estimated to have occurred during the Paleogene, 40.1 Mya (95% HPD: 19.1±64.3 Mya) (Fig 4, Paleocene to early Miocene). Since the origin of angiosperm plants during the Triassic, the most favorable climatic conditions for evergreen tropical vegetation in South America seems to have been during the early to middle Eocene [47]. There were two particularly noteworthy intense warming events in the Cenozoic: the short, PaleoceneEocene Thermal Maximum (PETM) beginning at 55.8 Ma and lasting for approximately 200-ka, and the longer, Early Eocene Climatic Optimum (EECO) lasting 2±4-Ma, when tropical temperatures reached *32±34ÊC [48±50]. The peak of the EECO occurred from 53 to 50 Ma as the culmination of a prolonged period of global warming and climatic change [51]. It was accompanied by substantial shifts in greenhouse gas concentrations, global temperatures, and precipitation patterns, as well as floral and faunal biogeographies. Reconstructions of terrestrial climatic and environmental conditions, based on data from the Southern Hemisphere, suggest that the EECO was marked by peak period of carbon isotope enrichment (up to 5% higher), increased mean annual temperature (up to 6ÊC higher), and increased mean annual precipitation (up to 500 mm yr ±1 higher) [52]. The increase in temperature has been associated with a significant increase in tropical plant diversity (*30%) [48,53,54] correlated with an Eocene radiation of terrestrial biota including arthropods such as leaf-cutter ants [55] evidenced a rich tropical rain forest in South America at that time. In Brazil, a Tropical Rainforest seems to have been the dominant biome during the Paleocene/Eocene according to fossil records from Brazilian deposits in Ipixuna/Para State, Maria Farinha/Pernambuco State and Bacia Itaboraõ  /Rio de Janeiro State [56] and the Olhos D'A  gua cave was likely to be surrounded by tropical rainforests during those periods. Thus, we can hypothesize that the range of Kimulidae was much more widespread in eastern Brazil during the Eocene, specifically the most recent common ancestor of the + !n clade. During the Oligocene to Miocene, cooler and drier climates caused a retraction of rainforest biomes while savannahs (e.g., Cerrado) and other less humid biomes flourished in South America [57]. This would have resulted in the constriction of ancestral ranges of biota that were restricted to warm, humid habitats. Thus, it appears that !n is a product of relictualism best explained by an Eocene radiation of Kimulidae in South America in which a single troglobitic lineage inhabiting the climatically stable, aphotic habitat inside the Olhos d'A  gua cave survived while the closest epigean relatives were driven to extinction by the changing climatic and environmental conditions surrounding the cave. The finding of this new Brazilian troglobite lends further support to the hypothesis that the Olhos d'A  gua cave acted as a stable refugium for myriad fauna, including this ancient lineage of Opiliones, and serves as an exemplary case of a hipogean habitat that represents a "museum" of biodiversity preservation (sensu Stebbins [58]) similar to the Australian Wet Tropics for Cyphophtalmi harvestmen [59] and Western and Central African forests for Ricinulei [60]. Systematic account Order OPILIONES Sundevall, 1833 Suborder LANIATORES Thorell, 1876 Family KIMULIDAE Pe  rez-Gonza  lez, Kury and Alonso-Zarazaga Pe  rez-Gonza  lez and Kury 2007 Type genus. Goodnight and Goodnight, 1942. Included genera. #n Gonza  lez-Sponga, 1998, Goodnight and Goodnight, 1942, " Avram, 1973, " Roewer, 1949, Roewer, 1949 and !n Pe  rez-Gonza  lez, Monte and Bichuette, gen. nov. (See previous family composition in [61]). A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 16 / 39

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Excluded genera (herein transferred to Zalmoxidae). $n Srensen, 1932, Kury and Alonso-Zarazaga, 2011, Mello-Leito, 1935 and Mello-Leito, 1933. n %&&'&( " )n( r r **& +* n*$#,-./,$0,12233$#/# r414/2//5r/ Type species. !n sp. nov. Included species. monotypic. Etymology. The genus name is a combination of "relicto"(from Latin n, past participle of 6 'leave behind'), referring to something that has survived from an earlier period or in a primitive form, and "" indicating its relationship to , the sister genus. Gender masculine. Comparative diagnosis. Individuals of !n gen. nov. are the smallest Kimulidae (1±1.1 mm body length) sharing the small size (less than 2 mm) with Roewer, 1949 and #n Gonza  lez-Sponga, 1998, whereas a group of `large-bodied kimulids' consists of Goodnight and Goodnight, 1942, " Avram, 1973 and " Roewer, 1949 (median 5.3 mm body length, ranging from 3.06±6.9 mm). Penis morphology of !n gen. nov. resembles that of but clearly differs from this and all other kimulid genera by the following characteristics: with the lamina ventralis partially surrounding the glans, latero-dorsally with three pairs of huge, wide and flattened macrosetae, conductors laminar expanded apically (hammer-like), wide rounded stylus surrounded by a thin ring-like parastylar collar. External morphology is similar to and together they differ from other kimulids by the broad base of the ocular tubercle (ocularium); the thick and massive spiniform apophyses on the antero-lateral border of carapace; and the mesotergal scutum with vestigial/incomplete sulci, except a deep, well-marked sulcus I and a shallower sulcus V. Additionally, in males of !n gen. nov. and , all mesotergal areas are approximately the same width (or slightly wider at areas IV±V) whereas in the `large-bodied kimulids' the widest part of the mesotergum is at the level of areas I±II. The other tiny kimulid, #n, also has the wider mesotergal portion at the level of areas IV±V, but it clearly differs from !n/ by the absence of an enlarged and armed femur IV, the absence of the strong spiniform apophyses on the antero-lateral border of carapace, the absence of armature on the free tergites and the morphology of the ocularium. !n gen. nov. differs from by the following external features: bell-shaped scutum magnum with the carapace wider relative to the mesotergum; posterior ocularium region of the carapace armed with two obtuse granulated setiferous tubercles; mesotergal areas with low, wide and blunt median granulated setiferous tubercles; femur IV less swollen; and troglomorphic features including anophthalmia and loss of pigmentation in the cuticle. Details of the exomorphological characteristics are provided in the species description below. Remark: !n gen. nov. and are closely related, but have several important exomorphological differences. These differences do not single-handedly justify the erection of another monotypic genus, and based on these data !n could be interpreted as a highly modified troglomorphic member of . Thus, in addition to exomorphology, our decision to erect a new genus is supported by several remarkable differences in male genitalia including: i) glans partially surrounded by lamina ventralis, whereas in , glans fully surrounded by lamina ventralis; ii) lamina ventralis contiguous instead of separated into two halves; iii) lamina ventralis laterally flat rather than strongly convex; iv) three pairs of macrosetae, rather than four; v) circumpenial concave fold absent in !n, but strongly developed in and located below the group of macrosetae; vi) conductors apically hammer-like instead of apically rounded; vii) two small pointed setae located ventrally, instead of laterally; viii) tubular stylus with a rounded tip instead of an enlarged tip. The gross A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 17 / 39

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differences in the penial groundplan between !n and are of the same magnitude as those which separate other kimulid genera such as , " and " . Furthermore, the intraspecific variation observed in the external and genital morphology between geographically isolated populations of n may correspond to a species complex, but that is beyond the scope of the present contribution and should be addressed in a future work. Distribution. Endemic to Peruac Ëu Caves National Park, Minas Gerais State, Brazil. n %&&'&, " )n, . . ::& .: n:--3),-)1124$311-.r55 r.0)1#2 (Figs 7±17). Type material. One male holotype (LES/UFSCar 0011188) from Olhos d'A Â gua cave [15Ê 6'49.32"S, 44Ê10'10.56"W], Peruac Ëu Caves National Park, Itacarambi, Minas Gerais State, Brazil, 24.x.2015, Monte, B.G.O., Zepon, T.; three female paratypes (LES/UFSCar 0011187), same data as holotype; one male paratype (LES/UFSCar 0011189, SEM voucher), same data as holotype; one female paratype (LES/UFSCar 0011190, SEM voucher), same data as holotype; one female paratype (MZUSP), same data as holotype; one male paratype (MACN) from Olhos d' A Â gua cave [15Ê 6'49.32"S, 44Ê10'10.56"W], Peruac Ëu Caves National Park, Itacarambi, Minas Gerais State, Brazil, 26.viii.2014, Monte, B.G.O., Bolfarini, M.P. Etymology. The species epithet is used as a noun in apposition. It refers to Galadriel (known as the Lady of Light), a character from J.R.R. Tolkien's famous novel "The Lord of the Rings". This name was chosen to signify the importance of such a species which sheds light on the poorly understood evolutionary and biogeographic history of Kimulidae, particularly their ancestral distribution in South America. Diagnosis. See diagnosis of the genus. Description. Males (Figs 7±14,15A and 15B). Body measurements in Table 5. Entire body finely granulated (e.g. Fig 7). Dorsum: n , bell-shaped with the mesotergal areas of approximately the same width, but slightly wider at the level of areas IV±V. Posterior margin of the n slightly convex. Carapace relatively wide compared to mesotergum (Ratio of mesotergum maximum width to carapace maximum width = 1.33). Carapace finely granulated, with the anterior margin slightly concave, cheliceral sockets not marked (Fig 7). Carapace in lateral view with a posterior ocularium region convex and armed with two obtuse granulated setiferous tubercles (Figs 7B and 8A), sulcus I well-marked. Antero-lateral border of the carapace armed with three acuminate strong tubercles (Fig 7B). Massive ocularium, granulated, terminating in a straight spiniform apophysis pointing anteriorly and with a broad and thick base (Fig 8B), and triangular in frontal view (Fig 8E). Eyes vestigial, apparently lacking the retina and cornea, with varying degree of degeneration, sometimes modified into a pointed tubercle (Figs 7B and 8B). Mesotergal scutum with five strongly convex areas (Figs 7D and 8A). Area I longer (along anterior-posterior axis) than remaining areas. Sulci between mesotergal areas I±IV incomplete, with a shallow, widened U-shaped line in sulci II and III (Fig 7D). Area I with a median region slightly elevated, covered with several low setiferous tubercles. Area II±IV with a median pair of low setiferous tubercles, larger on area IV. Area V with a transverse row of three low setiferous tubercles equal in size to those on area IV (Fig 7A and 7D). Free tergites each with one transverse row of low setiferous tubercles (Figs 7A, 7D, 8A and 8C). Ozopore region with well-marked descending, vertical and lateral channels (sensu Gnaspini and Rodrigues [62]) (Fig 8D). Coxa IV barely visible in dorsal view, terminating adjacent to sulcus III, with a prominent granulated obtuse setiferous tubercle at the prolaterodorsal surface, near the mesotergal n border (Fig 7A and 7C). Venter: free sternites each with a transverse row of prominent acute setiferous tubercles, larger medially (Fig 10A and 10B). Anal operculum covered by many low, robust setiferous tubercles of the same size as A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 18 / 39

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Fig 7. Relictopiolu s galadriel gen. nov., sp. nov. male paratype (LES/UFS Car 0011189), dorsal view. (A) Habitus. (B) Detail of carapace and chelicerae. (C) Detail of left coxa IV. (D) Detail of mesoterga l scute. A I-V: areas I to V; Cx IV: coxa IV; S II-V: sulci II to V. https://doi.o rg/10.1371/j ournal.pone .0187919.g007 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 19 / 39

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Fig 8. Relictopio lus galadriel gen. nov., sp. nov. male paratype (LES/UFS Car 001118 9). (A) Habitus lateral view. (B) Detail of ocularium, lateral view. (C) Detail of right coxa IV, lateral view. (D) Antero-late ral carapace showing the ozopore region. (E) habitus latero-fron tal view. (F) Detail of epistome, frontal view. Cx IV: coxa IV; Oz: ozopore; : epistome. https://do i.org/10.1371/j ournal.pone .0187919.g00 8 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 20 / 39

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those of the free tergites (Figs 8C and 10B). Coxa IV somewhat rounded, almost as wide as long, with several low wide setiferous tubercles (Fig 10A). Spiracles mostly concealed by coxa IV (Fig 10C and 10D). Epistome: epistome with sulcus well marked. Post-sulcal epistome wider than tall with a medial groove dividing the post-sulcal epistome into two convexes domes. Basal pre-sulcal epistome wide and short, almost triangular. Pre-sulcal epistome process long and triangular without median constriction (Fig 8F). Chelicera: basichelicerite unarmed with a well-marked rounded bulla. Cheliceral hand unarmed, normal, neither swollen nor hypertelic, covered with several sensilla. Mobile finger with uniform rounded teeth (Fig 9). Pedipalp: raptorial morphotype (sensu Wolff et al. [63]) (Fig 11A and 11C). Coxa short, unarmed, finely granulated. Trochanter globular, with one dorsal and two ventral small setiferous tubercle. Femur armed ventrally with one proximal and one medial major spines (i.e. stiff pointed bristles in highly elevated sockets, sensu Wolff et al. [63]) with one small pointed setiferous tubercle; dorsally with three small pointed setiferous tubercles and one subdistal-mesal major spine. Patella cylindrical, armed with one ventro-medial major spine on the mesal surface. Tibia armed ventrally with three ectal and three mesal major spines. Tarsus armed ventrally with three ectal and three mesal major spines (Fig 11A and 11C). All major spines possess very small and sparse microtrichia covering the distal half (Fig 11B). Legs (Fig Fig 9. Relictopi olus galadri el gen. nov., sp. nov. male paratype (LES/UFSCa r 0011189). Right cheliceral hand, frontal view. https://d oi.org/10.1371/j ournal.pon e.0187919.g0 09 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 21 / 39

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12): leg measurements in Table 5. Cuticle of legs is scale-like and granulated except on calcaneus and tarsus. Calcaneus restricted to the distal portion of legs. Femur I with a ventral row of four marked setiferous tubercles. Leg IV sexually dimorphic, males with a strong pointed tubercle in the ventro-distal portion of the trochanter (Fig 10A) and a moderately swollen femur with two ventral rows of low setiferous tubercles (Fig 12D). Tarsal formula: 3(2):4 (2):4:5. Male genitalia (Figs 13 and 14): Pars distalis swollen separated from the pars basalis by slight constriction. Pars distalis with the lamina ventralis flat and slender, partially surrounding the glans, latero-dorsally with three pairs of huge, wide and flattened macrosetae and ventrally with two small pointed setae. Glans with a pair of laminar conductors expanded apically (hammer-like) and a wide stylus surrounded by a thin ring-like parastylar collar. Remark: Fig 10. Relictopio lus galadriel gen. nov., sp. nov. male paratype (LES/ UFSCar 001118 9), ventral view. (A) Habitus. (B) Detail of free sternites and anal operculum . (C) Detail of spiracle area on right side. (D) Detail of concealed spiracle on right side. All images are oriente d such that the anterior of the animal is at the top. https://do i.org/10.1371/j ournal.pone .0187919.g01 0 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 22 / 39

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the general groundplan of male genitalia resemble those of the close relative n (Fig 18B, 18C and 18F±18H), but exibits clear differences in the pars distalis (e.g. lamina ventralis, macrosetae and conductors morphologies). Furthermore, the external morphology of !n is most similar to that of n (Fig 18A, 18D and 18E) compared to other kimulids. See !n "comparative diagnosis" above for details. Females (Figs 15C; 16 and 17). Body measurements in Table 5. Similar in appearance to the males but without the swollen femur IV and with more tubercles in mesotergal areas (Fig 16). Color (in alcohol). The color (both sexes) of the whole body is Brilliant Greenish Yellow (98). Legs are more transparent at apices, tarsus Pale Greenish Yellow (104), trochanter, femur, patella and tibia Light Greenish Yellow (101) (Fig 15). Distribution. Known only from the type locality. Natural history. Specimens were collected during five different expeditions to the Olhos d'A Â gua cave, usually far from the entrance, about 1200 m from the resurgence (Fig 2A). Only one individual was collected nearer to the entrance, about 450 meters from the resurgence (Fig 2B). All individuals were observed and captured on the cave walls, in the largest galleries (3 m wide and 10 m high) (cave cross section I in Fig 2). Temperature (~28ÊC) and humidity (90% of RH) tend to be constant in the localities where the harvestmen were observed. Some individuals were observed in close proximity to two other troglobitic arachnids from Olhos d'A Â gua cave: the opilionid Pinto-da-Rocha, 1997 and the amblypygid Baptista and Giupponi, 2003. Other fauna observed in the galleries where !n occurs includes nectarivorous and hematophagous bats, millipedes (Polydesmida), a troglobitic cricket (n Bolfarini and Bichuette, 2015), coleopterans (families Cholevidae and Carabidae) and arachnids of the order Palpigradi. Spurious Kimulidae: justification of generic exclusions and nomenclatural implications. $n Srensen, 1932 $n [64]: 248; [65]: 91; [66]: 138; [67]: 40; [68]: 110; [69]: 62; [46]: 211 [type species: $n n Srensen, 1932, by monotypy, (examined)]. [70]: 5; [71]: 232; [46]: 249 [type specie: 7 Caporiacco, 1951, by monotypy, (not examined)]. New synonymy. Fig 11. Relictopio lus galadriel gen. nov., sp. nov. male paratype (LES/ UFSCar 001118 9). (A) Right pedipalp, ectal view. (B) Detail of major spine indicated by . (C) Left pedipalp, mesal view. : major spine. https://do i.org/10.1371/j ournal.pone .0187919.g01 1 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 23 / 39

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The genus $n is currently considered a member of Kimulidae [61] but the examination of two males syntypes of the type species $n n Srensen, 1932 (repository ZMUC) revealed that the exomorphology (Fig 19A±19C) does not match with the familial characteristics as defined by Pe  rez-Gonza  lez and Kury [72]; therefore, $n is herein transferred to Zalmoxidae Srensen, 1886, new family allocation. Moreover, the morphology of one male syntype of $. n was highly congruent with the drawings of nn Gonzalez-Sponga, 1987, including diagnostic characteristics such as the particular armature of trochanter IV with two huge apophyses (see [71]: 241, figs. 283±289 and Fig 19C). Type localities for these two species (Venezuela: Carabobo, Fig 12. Relictopio lus galadriel gen. nov., sp. nov. male paratype (LES/ UFSCar 001118 9), right legs, retrolater al views. (A) Leg I. (B) Leg II. (C) Leg III. (D) Leg IV. https://do i.org/10.1371/j ournal.pone .0187919.g01 2 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 24 / 39

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Fig 13. Relictopi olus galadri el gen. nov., sp. nov. male paratype (LES/UFSCa r 0011189), male genitalia. (A) Dorsal view. (B) Detail of pars distalis, dorsal view. (C) Detail of pars distalis, lateral view. (D) Detail of glans, dorsal view. (E) Detail of glans, lateral view. (F) Detail of pars distalis, ventral view. (G) Detail of pars distalis, apical view. (H) Detail of glans, apical view. Abbreviat ions, C: constriction; Co: conductor ; G: glans; Fm: flattened macrosetae; Lv: lamina ventrali s; Pc: parastylar collar; Pb: pars basalis; Pd: pars distalis; Ps: pointed setae; S: stylus. : ductus ejaculatorius opening. https://do i.org/10.1371/j ournal.pon e.0187919.g0 13 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 25 / 39

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Las Trincheras, for $n n and Venezuela: Carabobo, Puerto Cabello, San Esteban, 200 m. for nn) are relatively close (~15 km). Therefore, we propose that $n n Srensen, 1932 is a senior synonym of nn Gonzalez-Sponga, 1987, new synonymy. The genus was established by Caporiacco [70] to accommodate the new species 7 Caporiacco, 1951. The species was described using a type series composed of four females and one juvenile from two different Venezuelan localities: El Junquito, D.F. and Rancho Grande (currently National Park Henri Pittier), Aragua. GonzalezSponga [71] treated these as different species; he restricted 7 to the type locality (El Junquito) and described a new species, Gonza  lez-Sponga, 1987, for the species living in Rancho Grande. Gonza  lez-Sponga [71] also described 11 more species under . Comparing the descriptions and drawings of Caporiacco [70] and Gonza  lez-Sponga [71] we agree that the current 13 species comprising the genus are congeneric based on the congruence of morphological and biogeographical data. In his revisionary work, Gonzalez-Sponga [71] unfortunately did not examine any of Srensen's types causing him to overlook the synonymy between $n and . Following the priority principle [73] we consider $n Srensen, 1932 as a senior synonym of Caporiacco, 1951, new synonymy. The proposed synonymy results in the following new combinations: $n (Gonza  lez-Sponga, 1987) new combination, $n (Gonza  lez-Sponga, 1987) new combination, $n nn (Gonza  lez-Sponga, 1987) new combination, $n nn (Gonza  lez-Sponga, 1987) new combination, $n n (Gonza  lez-Sponga, 1987) new combination, $n nnn (Gonza  lez-Sponga, 1987) new combination, $n n88n (Gonza  lez-Sponga, 1987) new combination, $n (Gonza  lez-Sponga, 1987) new combination, $n (Gonza  lez-Sponga, 1987) new combination, $n (Gonza  lez-Sponga, 1987) new combination, $n 7 (Caporiacco, 1951) new combination, $n Fig 14. Line drawings of male genitalia of Relictopiol us galadriel gen. nov., sp. nov. male paratype (LES/UFS Car 0011189 ). (A) Detail of pars distalis, lateral view. (B) Same, ventral view. (C) Same, dorsal view. https://doi.org/10 .1371/journal.p one.0187919. g014 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 26 / 39

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(Gonza  lez-Sponga, 1987) new combination, $n (Gonza  lez-Sponga, 1987) new combination. $ : Kury [46] erroneously stated that the type locality for $n n was "Venezuela, Distrito Federal, Las Trincheras, (10Ê33'N, 66Ê59'W)". Srensen [64] described the type locality as: "Patria: Venezuela. In the month of December 1891 at Las Trincheras CHR. LEVINSEN (LOFTING). . .", and the original label contains the same data with a more specific date (21±23 of December of 1891). Gonzalez-Sponga [71] stated in his Fig 15. Habitus of specime ns (in alcohol ) of Relictop iolus galadr iel gen. nov., sp. nov. (A) Male holotype (LES/UFS Car 0011188 ), dorsal view. (B) Same, lateral view. (C) Female paratype (LES/UFS Car 0011187), dorsal view. https://do i.org/10.1371/j ournal.pon e.0187919.g0 15 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 27 / 39

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Fig 16. Relictopiolu s galadriel gen. nov., sp. nov., female paratype (LES/UFS Car 0011190), dorsal view. (A) Habitus. (B) Detail of carapace and chelicera e. (C) Detail of right coxa IV. (D) Detail of mesoterga l scute. https://doi.org/10 .1371/journal.p one.0187919. g016 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 28 / 39

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Historical Background that Srensen, in 1891, worked on material collected by C. Levinsen in Las Trincheras, and determined this locality to be in the Carabobo State of Venezuela. Las Fig 17. Relictopio lus galadriel gen. nov., sp. nov., female paratype (LES/ UFSCar 001119 0), lateral view. (A) Habitus. (B) Detail of posterior opisthosom a. (C) Detail of ocularium. https://do i.org/10.1371/j ournal.pone .0187919.g01 7 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 29 / 39

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Trincheras is a small town near the city of Valencia in the State of Carabobo (10Ê18'22.64"N, 68Ê 5'19.68"W, Google Earth). Table 5. Somatic and appendicular measureme nts for Relictopiol us galadriel gen. nov. et sp. nov. Relicito piolus galadri el gen. nov. et sp. nov. Male holotype (LES/U FSCar 0011188) Male paratype (MACN) Female paratype (LES/U FSCar 0011187) Female paratype (LES/U FSCar 0011187 ) Female paratype (LES/U FSCar 0011187 ) Female paratype (MZUSP) total body length 1.10 1.10 1.10 1.04 1.11 1.06 scutum magnum length 1.00 0.90 1.00 0.93 0.96 0.94 carapace length 0.40 0.40 0.40 0.38 0.39 0.38 carapace maximum width 0.60 0.60 0.60 0.55 0.55 0.63 mesoterga l scute maximum width 0.80 0.80 0.80 0.81 0.83 0.86 tr 0.10 0.10 0.10 0.10 0.12 0.09 fe 0.40 0.40 0.40 0.35 0.38 0.39 Pedipalp pa 0.20 0.20 0.20 0.17 0.20 0.19 ti 0.30 0.30 0.30 0.25 0.30 0.26 ta 0.30 0.20 0.20 0.23 0.25 0.24 Total 5.20 5.00 5.10 4.82 5.09 5.05 tr 0.10 0.10 0.10 0.09 0.10 0.10 fe 0.50 0.50 0.50 0.50 0.49 0.51 pa 0.20 0.20 0.20 0.22 0.26 0.24 Leg I ti 0.30 0.40 0.30 0.32 0.34 0.33 me 0.40 0.50 0.40 0.39 0.46 0.43 ta 0.50 0.50 0.50 0.41 0.44 0.43 Total 2.00 2.20 2.00 1.92 2.10 2.05 tr 0.10 0.20 0.10 0.10 0.12 0.13 fe 0.70 0.60 0.60 0.68 0.74 0.72 pa 0.30 0.20 0.30 0.32 0.33 0.31 Leg II ti 0.60 0.50 0.60 0.56 0.60 0.56 me 0.50 0.60 0.50 0.45 0.50 0.50 ta 1.00 0.50 1.00 0.96 1.05 1.00 Total 3.20 2.60 3.10 3.07 3.34 3.21 tr 0.10 0.20 0.10 0.12 0.10 0.09 fe 0.60 0.60 0.60 0.56 0.59 0.54 pa 0.20 0.20 0.20 0.19 0.20 0.21 Leg III ti 0.50 0.50 0.40 0.40 0.47 0.41 me 0.60 0.60 0.60 0.55 0.61 0.54 ta 0.50 0.40 0.40 0.40 0.45 0.38 Total 2.50 2.50 2.30 2.22 2.42 2.17 tr 0.20 0.20 0.10 0.11 0.15 0.14 fe 0.80 0.80 0.70 0.72 0.73 0.71 pa 0.40 0.30 0.30 0.29 0.33 0.28 leg IV ti 0.50 0.70 0.60 0.59 0.62 0.59 me 0.80 0.80 0.80 0.71 0.78 0.72 ta 0.50 0.60 0.50 0.50 0.55 0.50 Total 3.20 3.40 3.00 2.92 3.16 2.95 Abbrevia tions: tr = trochante r, fe = femur, pa = patella, ti = tibia, ta = tarsus. https://do i.org/10.1371/j ournal.pone .0187919.t005 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 30 / 39

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Fig 18. Tegipi olus pachypus Roewer , 1949, (A) male holotype and (B,C) male paratype (SMF 9906896, Tegipio , Pernambuco State, Brazil), (D-H) male (IBSP 7071, Murici, Alagoas State, Brazil). (A) Habitus, dorso-latera l view. (B) Penis, dorsal view. (C) same, lateral view. (D) Habitus, dorsal view. (E) same, lateral view. (F) Penis, dorsal view. (G) Same, lateral view. (H) Same, apical view. Abbreviation s, Ccf: circum plenial concav e fold; Co: conductor ; Fm: flattened macroseta e; G: glans; Lv: lamina ventralis; Pc: parastylar collar; S: stylus; : ductus ejaculato rius opening; black arrows indicate the strongly convex lateral portio n of lamina ventralis. https:// doi.org/10.137 1/journal.pone. 0187919.g018 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 31 / 39

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Fig 19. Spurious Kimulida e. (A-C) Acanthomi nua tricarinat a S¡rensen, 1932 (male syntype , ZMUC). (A) Habitus, dorsal view. (B) Detail of right trochanter IV, ventral view. (C) Habitus, lateral view. (D) Euminua brevitars a S¡rensen, 1932 (male syntype , ZMUC), habitus, dorsal view. (E) Euminua longitars a S¡rensen, 1932 (male syntype , ZMUC), habitus, dorsal view. (F-I) Euminu a convolvulus S¡rensen, 1932. (F) Habitus, dorsal view (male syntype, ZMUC). (G-I) Male genitalia (male syntype, SMF). (G) Lateral view. (H) Dorsal view. (I) Ventral view. https://doi.o rg/10.1371/j ournal.pone .0187919.g019 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 32 / 39

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Kury and Alonso-Zarazaga, 2011 [64]:239; [65]: 92; [66]: 138; [68]: 110; [69]:62; [71]: 205, [46]: 211. Unavailable name (see [74]:59). [74]: 59 [type species: Srensen, 1932, by original designation (examined)]. The genus Kury and Alonso-Zarazaga, 2011, was until now a member of Kimulidae [61]. The examination of three syntypes of the type species Srensen, 1932, (repository ZMUC) revealed that the species exhibits a peculiar sexually dimorphic character: in males, leg IV is much longer than that of females (Fig 19D). This character is common in several genera and species of Zalmoxidae (e.g. [75]) several of which are recorded for Venezuela (e.g. n (Gonza  lez-Sponga, 1987), n9 Gonza  lezSponga, 1987, 7 Caporiacco, 1951, Gonza  lezSponga, 1987 and :n 8 Gonza  lez-Sponga, 1987). Therefore, is herein transferred to Zalmoxidae Srensen, 1886, new family allocation. Mello-Leito, 1935 [65]: 92; [66]: 138; [68]: 110; [46]:211 (type Srensen, 1932, by original designation, (examined)]. The genus Mello-Leito, 1935 was, until now, a member of Kimulidae [61]. The examination of 14 syntypes of the type species Srensen, 1932, (repository ZMUC) revealed that the exomorphology (Fig 19E) does not match with the familiar characteristics as defined by Pe  rez-Gonza  lez and Kury [72] and the illustrations of the male genitalia by Srensen ([64], Fig 5A±5C) show a form that is clearly related to other Zalmoxidae with a pergula and rutrum (as defined by Kury and Pe  rez-Gonza  lez [76]). Moreover, males of this species exhibit an elongated leg IV (Fig 19E), a sexually dimorphic character also described for (see above). Therefore, is herein transferred to Zalmoxidae Srensen, 1886, new family allocation. Mello-Leito, 1933 [77]: 101; [66]: 138; [78]: 45; [71]: 205; [46]:213 [type species: n Srensen, 1932, by original designation, (examined)]. The genus Mello-Leito, 1933 was assigned to Kimulidae [61] but the examination of 5 syntypes of the type species n Srensen, 1932, (ZMUC: 4 syntypes; SMF: 1 syntype) revealed that the exomorphology (Fig 19F) does not match with the familiar characteristics as defined by Pe  rez-Gonza  lez and Kury [72] and the male genitalia, with a pergula and rutrum, demonstrate their close relationship with Zalmoxidae (as defined by Kury and Pe  rez-Gonza  lez [76]) (Fig 19G±19I). Therefore, is herein transferred to Zalmoxidae Srensen, 1886, new family allocation. $ : Kury [46] erroneously stated that the type locality for n (Srensen, 1932) was "Venezuela, Distrito Federal, Las Trincheras, (10Ê33'N, 66Ê 59'W)". Srensen [64] described the type locality as: "Patria: Venezuela. MEINERT collected 2 females and 5 males at Las Trincheras November 5th, 1891. . .", and the original label contains the same data. Gonzalez-Sponga [71] stated that the locality "Las Trincheras" is located in Carabobo State. Las Trincheras is a small town near the city of Valencia in the State of Carabobo (10Ê18'22.64"N, 68Ê 5'19.68"W, Google Earth). Implications for cave conservation Some authors consider caves as hotspots of subterranean biodiversity if they contain 20 or more obligate subterranean species, a rather arbitrary number proposed by Culver and Sket [79]. Presently, there are 38 caves in the world that fall under this definition of a subterranean A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 33 / 39

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hotspot [79±81]). Taking a more comprehensive approach, Trajano et al. [82] adopted the concept of ªspots of high diversity of troglobitesº based not only on taxonomic richness, but also considering phylogenetic and genetic diversity. Thus, the Olhos d'A Â gua cave, with at least 11 troglobites, as well as several other caves in Brazil, are considered important locales for subterranean diversity [82]. The Olhos d'A Â gua cave contains taxa that represent phylogenetically and biogeographically important lineages, as well as genetic diversity, the latter based on the degree of specialization of individual species, that is, accumulation of autapomorphies. The presence of !n in the Olhos d'A Â gua is evidence that the cave provided a stable refugium for millions of years and acted as a "museum" of biodiversity. This indicates the need to consider some measure of phylogenetic and/or biogeographic relevance as additional criteria for ranking the importance of the different "spots of high diversity of troglobites" as defined by Trajano et al. [82]. In a separate study of this cave, data indicate that the Olhos d'A Â gua cave is ecologically important based on its relatively high values of Taxonomic Distinctness (TD) (Monte and Bichuette pers. obs.). The extraordinary diversity (i.e., morphological, phylogenetic, genetic and functional diversity) of the Olhos d'A Â gua cave makes it a high conservation priority, but there are several challenges that must be overcome to ensure the continual protection of this fragile habitat and its unique biota. First, the cave's resurgence, which serves as the only access point, is located outside of the PCNP boundaries, and therefore is not legally protected by the National Parks system in Brazil. Additionally, the protection of cave ecosystems, such as the Olhos d'A Â gua cave, requires synergistic conservation efforts to protect connected ecosystems, including lotic and terrestrial ecosystems, which support life in the cave. The Olhos d'A Â gua cave community is dependent upon allochthonous resources, or energy sources that are derived from outside of the cave, that are introduced to the cave by natural flash flooding in the region. Decreasing the input of organic matter can threaten the ecological functioning of a subterranean ecosystem by disrupting the trophic dynamics. Thus, flash floods are crucial to the maintenance of the cave community because they distribute organic matter 5000 m into the cave, thus providing energy sources that constitute a primary trophic level within the cave ecosystem. However, the number of flash flooding events has dramatically decreased over the last two decades, and this scarcity of floods has led to negative impacts on cave fauna, such as the significant decline in the population of the cave catfish nn n Trajano and de Pinna, 1996 between 1994 and 2007 [83]. Another threat to the Olhos d'A Â gua cave community is the increased use of groundwater for agricultural purposes in the region, accompanied by the construction of several dams upstream of the sinkhole and inside the cave to store water for use during the dry season (B.G.O.M and M.E.B., pers. obs.). Given that the Olhos d'A Â gua cave system may be under intense ecological stress due to the scarcity of flash floods, the cavernicolous fauna is at risk of becoming endangered or extinct and conservation actions are urgently required in order to protect this delicate, valuable habitat and avoid causing irreversible damage to its unique biota. !n is the first relictual species discovered in the PCNP region and represents a key taxon for improving our understanding of the evolutionary and biogeographic history of an ancient lineage of harvestmen. Given that this troglobitic harvestman is a relictual species that exhibits low population density, and is endemic to a single cave that is under ecological stress due to global climate change as well as anthropogenic changes to the local environment, this species is a strong candidate for inclusion on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Its inclusion on the Red List would establish a means for governmental authorities and the scientific community to make informed decisions and implement concrete conservation actions to prevent the loss of biodiversity in this cave. A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 34 / 39

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Supporting information S1 Fig. Fully-resolved phylogenetic tree obtained by Bayesian inference analysis of the complete concatenated dataset, conducted in MrBayes. Support values at nodes represent posterior probabilities. (PDF) S2 Fig. Chronogram obtained by Bayesian inference analysis of the complete concatenated dataset, conducted in BEAST. Support values at nodes represent posterior probabilities and blue bars represent the 95% Highest Posterior Densities around divergence time estimates. (PDF) S3 Fig. Fully-resolved phylogenetic tree obtained by Bayesian inference analysis of the complete concatenated dataset including COI and H3 third codon positions, conducted in MrBayes. Support values at nodes represent posterior probabilities. (PDF) S4 Fig. Chronogram obtained by Bayesian inference analysis of the complete concatenated dataset including COI and H3 third codon positions, conducted in BEAST. Support values at nodes represent posterior probabilities and blue bars represent the 95% Highest Posterior Densities around divergence time estimates. (PDF) Acknowledgmen ts We are indebted with J.E. Gallo, M. P. Bolfarini and T. Zepon for help in the field. We would like to thank A.M.P.M. Dias, coordinator of the National Institute of Science and Technology of the Hymenoptera Parasitoids from Brazilian Southeast Region (INCT Hympar Sudeste), for permission to use the scanning electron microscope (SEM) and stereomicroscope; L.B.D.R. Fernandes from UFSCar, for taking the SEM and stereomicroscope images of !. . The following research projects kindly provided specimens for inclusion in the molecular analyses: PANCODING (PI's: Joan Pons, Miquel Arnedo), CarBio (PI's Ingi Agnarsson, Greta Binford). APG is especially grateful to Peter Ja Èger and Julia Altmann (SMF) for their very kind hospitality during his visit to the SMF and for providing access to Roewer's collection. The following curators kindly loaned specimens for this study: Nikolaj Scharff (ZMUC), Adriano Kury (MNRJ), E  lisson Bezerra Lima (CHNUFPI), Antonio D. Brescovit (IBSP). We acknowledge Leonardo Sousa Carvalho for collecting specimens and for arranging the CHNUFPI loan. Põ  o Colmenares and Andre  s Garcõ  a kindly shared unpublished information about Kimulidae records in South America. We want to thank Willians Porto for taking pictures of Srensen's types and n. Maria Judite Garcia kindly shared bibliographic information about South American Paleogene floras. We are indebted to A. Kury for providing valuable bibliographic information through his altruistic Omnipaper Project (http://www.museunacio nal.ufrj.br/mndi/Aracnologia/ pdfliteratura/pdfs%20opilione s.htm). The cartographic map of Olhos d'A  gua cave was a courtesy of the Bambuõ  Speleological Group. Ana Laura Carbajal de la Fuente kindly helped to format the final draft of the manuscript. We want to thank Gonzalo Giribet, Darrell Ubick and one anonymous reviewer for their insightful comments and suggestions that improved the manuscript. Author Contributions Conceptualization: Abel Pe  rez-Gonza  lez, F. Sara Ceccarelli, Maria E. Bichuette. Formal analysis: F. Sara Ceccarelli. A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 35 / 39

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Funding acquisition: Abel Pe  rez-Gonza  lez, Maria E. Bichuette. Investigation: Abel Pe  rez-Gonza  lez, F. Sara Ceccarelli, Bruno G. O. Monte. Supervision: Abel Pe  rez-Gonza  lez, Maria E. Bichuette. Visualization: Abel Pe  rez-Gonza  lez, F. Sara Ceccarelli, Bruno G. O. Monte. Writing ± original draft: Abel Pe  rez-Gonza  lez, F. Sara Ceccarelli, Bruno G. O. Monte, Daniel N. Proud, Ma  rcio Bernardino DaSilva, Maria E. Bichuette. Writing ± review & editing: Abel Pe  rez-Gonza  lez, F. Sara Ceccarelli, Bruno G. O. Monte, Daniel N. Proud, Ma  rcio Bernardino DaSilva, Maria E. Bichuette. References 1. Culver DC. Karst environ ment. Z Geomorphol . 2016; 60(2): 103±11 7. https://doi.or g/10.1127/ zfg_ suppl/201 6/00306 2. Poulson TL., White WB. The cave environm ent. Science. 1969; 65(3897): 971±981. https://doi.or g/10. 1126/scien ce.165.38 97.971 PMID: 17791021 3. Gibert J, Deharveng L. Subterran ean Ecosyste ms: A Truncated Functiona l Biodive rsity. BioScience. 2002; 52(6): 473±481. https://doi.or g/10.1641/ 0006-3568( 2002)052[04 73:SEATFB]2 .0.CO;2 4. Rambla M, Juberthie C. Opiliones. In: Juberthi e C, Decu V, editors. Encyclopae dia Biospeo logica: Socie  te  de Biospe  ologie, Moulis-B oucarest; 1994. pp. 215±230. 5. Trajano E, Carvalho MR. Towards a biologic ally meaning ful classification of subterr anean organism s: a critical analysis of the Schiner-R acovitza system from a historical perspective , difficultie s of its application and implication s for conserva tion. Subterr Biol. 2017; 22: 1±26. https:// doi.org/10.38 97/subtbio l.22. 9759 6. Cruz-Lo  pez JA, Proud DN, Pe  rez-Gonza  lez A. When troglomorphi sm dupes taxonomists : morphology and molecules reveal the first pyramido pid harvestman (Arachnida , Opiliones , Pyramidopidae ) from the New World. Zool J Linn Soc Lond. 2016; 177(3): 602±620. https://d oi.org/10.111 1/zoj.1238 2 7. Hara MR, Pinto-da-Roch a R. A new species of Brazilian troglobitic harvestm an of the genus Iandumoema (Opilione s, Gonylept idae). Zootaxa. 2008; 1744: 50±58 8. Willemar t RH, Taques B. Morfologia e ecologia sensorial em aracnõ  deos troglo  bios: perspectiva s para a espeleo biologia brasileira. Rev Biol. 2013; 10(2): 46±51. 9. Trajano E, Bichuette ME. Diversit y of Brazilian subterranea n invertebrates, with a list of troglomorphi c taxa. Subterr Biol. 2009; 7: 1±16. 10. DaSilva MB, Pinto-da-R ocha R, Giribet G. Canga renatae, a new genus and species of Cyphophtha lmi from Brazilian Amazon caves (Opilione s: Neogoveida e). Zootaxa; 2508, 45±55. 11. Pilo  LB, Ko È hler HC. Do Vale do Peruac Ë u ao Smo Francisco: uma viagem ao interior da terra. In: Abequa editor. Congresso da Associac Ë mo Brasileira do Estudo do Quate rna  rio, 3: Roteiro de Excursmo. Belo Horizonte, Brasil: Imprensa Universita  ria da UFMG; 1991. pp. 57±73. 12. Ab'Saber AN. Os domõ  nios morfoclima  ticos na Ame  rica do Sul: primeira aproxima c Ë mo. Geomorfolog ia. 1st ed. Smo Paulo: Instituto de Geografia da Universidad e de Smo Paulo Press; 1977. 13. Peel MC, Finlayson BL, McMah on TA. Updated world map of the Ko È ppen-Gei ger climate classification, Hydrol Earth Syst Sci. 2007 Oct 11. pii: 1633-16 44(11). https:// doi.org/10.51 94/hess-1 1-1633-20 07 14. Ramos AM, Santos LAR, Fortes LTG. Normais Climato  logicas do Brasil 1961±1 990. 1st ed. Brasilia: Instituto Nacional de Meteorol ogia Press; 2009. 15. Kury AB, Orrico VGD. A new species of Lacronia Strand, 1942 from the highlands of Rio de Janeiro (Opilione s, Gonylept idae, Pachylinae). Rev Iber Aracno l. 2006; 13: 147±153. 16. Acosta LE, Pe  rez-Gonza  lez A, Tourinho AL. Methods and technique s of study: Methods for taxonomic study. In: Machado G, Pinto-da-R ocha R, Giribet G, editors. Harvestm en: the biology of Opiliones. Cambridge : Harvard University; 2007. pp. 494±505. 17. Sharma PP, Giribet G. The evolution ary and biogeograp hic history of the armoured harvestmen± Laniatores phyloge ny based on ten molecular markers, with the description of two new families of Opiliones (Arachnida ). Invertebr Syst. 2011; 25(2): 106±142. https://doi.or g/10.107 1/IS11002 18. Sharma PP, Giribet G. Out of the Neotropics : Late Cretaceous colonizatio n of Australasia by American arthropods. Proc R Soc Lond B Biol Sci. 2012; 279(1742) : 3501±3509. https://doi.or g/10.1098/rs pb. 2012.0675 PMID: 22628468 A new troglobite is evidence of the ancestral distribution of Kimulidae in South America PLOS ONE | https://doi.or g/10.137 1/journal.po ne.01879 19 November 30, 2017 36 / 39

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