Molecular Phylogenetics and Evolution 174 (2022) 107530 Available online 27 May 2022 1055-7903/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Huntsman spider phylogeny informs evolution of life history, egg sacs, and morphology Jacob A. Gorneau a,b,*, Cristina A. Rheims c, Corrie S. Moreau a,d, Linda S. Rayor a a Department of Entomology, Cornell University, Ithaca, NY 14853, USA b Institute for Biodiversity Science and Sustainability, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA 94118, USA c Laboratório de Coleções Zoológicas, Instituto Butantan, Av. Vital Brasil, 1500, 05503-900 São Paulo, SP, Brazil d Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA A R T I C L E I N F O Keywords: Sparassidae Molecular phylogenetics Social evolution Stochastic character mapping D-test Deleninae A B S T R A C T Huntsman spiders (Araneae: Sparassidae) are among the most speciose spider families, with a near-worldwide distribution, diverse habitats, equally diverse life histories, and five prolonged subsocial species. Previous mo- lecular phylogenies have focused on individual subfamilies or clades. Here, we provide a phylogenetic inference with broadened sampling from 37 genera and eight of the eleven sparassid subfamilies. We increased taxon sampling by including species not previously sequenced and most available data on GenBank of two mito- chondrial (COI, 16S rRNA) and two nuclear (H3, 28S rRNA) genes for a total of 262 ingroup taxa and nine outgroup taxa. Divergence dates were estimated using outgroup fossil taxa suggesting the sparassids evolved ~ 100 mya (stem age), while the clade containing all subfamilies except Sparianthinae evolved ~ 90 mya (stem age). Using a stochastic map approach with 40 species, this is the first sparassid phylogeny to incorporate extensive biology and life history data. Correlations of life history traits with solitary, subsocial, and prolonged subsocial behavior are examined using the D-test. Sparassid sociality is associated with life history traits that allow developing spiders to remain in their natal retreat longer (e.g., larger permanent retreats, plastered egg sacs, and ontogenetically delayed foraging), but is unrelated to body size or lifespan. Detailed morphological scoring of the endemic Australian subfamily Deleninae contextualizes existing molecular data, including in the Isopeda-Holconia-Isopedella complex. This study supports the monophyly of many major lineages, including for the first time, the Sparianthinae, but indicates multiple clades (Sparassinae and Eusparassinae) are paraphyletic and need further revision. 1. Introduction Huntsman spiders (Araneae: Sparassidae) comprise 1,319 species described from 91 genera (World Spider Catalog, 2022 23.0), making them the eleventh-most diverse family of spiders (see Fig. 1 for a snapshot of sparassid biodiversity, with a focus on individuals in this study). Although already quite diverse, it is estimated that at least 1,000 huntsman species remain to be described (Agnarsson et al., 2013a). Huntsmen have a near-worldwide distribution and are found in many climates and habitats, ranging from deserts to rainforests, lowlands to highlands, and in caves (Jäger, 2005a, Jäger, 2005b, Rheims, 2010a, Rheims, 2010b, Hsieh & Linsenmair, 2011, Moradmand et al., 2016). The monophyly of huntsman spiders is unquestioned morphologi- cally and has been corroborated by multiple morphological (Rheims, 2007a, Ramírez, 2014) and molecular phylogenetic inferences (Agnarsson & Rayor, 2013, Moradmand et al., 2014, Wheeler et al., 2017, Tong et al., 2019). Huntsman spiders were first described by Bertkau (1872), though use of the name Sparassidae was disputed until recently, with Heteropodidae and Eusparassidae also in use through the end of the 20th century (Jäger, 1999a). A conspicuous synapomorphy for the family is the presence of a trilobate membrane dorsally on the apex of the metatarsus, as proposed by Petrunkevitch (1928), but other synapomorphies identified by Ramírez (2014) include the apical indentation of claw tuft setae, membranous projections of the tarsi * Corresponding author at: Institute for Biodiversity Science and Sustainability, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA 94118, USA. E-mail addresses: jag482@cornell.edu, jgorneau@calacademy.org (J.A. Gorneau), carheims@gmail.com (C.A. Rheims), corrie.moreau@cornell.edu (C.S. Moreau), lsr1@cornell.edu (L.S. Rayor). Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev https://doi.org/10.1016/j.ympev.2022.107530 Received 29 July 2021; Received in revised form 17 April 2022; Accepted 6 May 2022 mailto:jag482@cornell.edu mailto:jgorneau@calacademy.org mailto:carheims@gmail.com mailto:corrie.moreau@cornell.edu mailto:lsr1@cornell.edu www.sciencedirect.com/science/journal/10557903 https://www.elsevier.com/locate/ympev https://doi.org/10.1016/j.ympev.2022.107530 https://doi.org/10.1016/j.ympev.2022.107530 https://doi.org/10.1016/j.ympev.2022.107530 http://crossmark.crossref.org/dialog/?doi=10.1016/j.ympev.2022.107530&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ Molecular Phylogenetics and Evolution 174 (2022) 107530 2 around claw tuft plates, and trichobothrial setae with smooth bases. Based on morphology and phylogenetic analysis, the placement of huntsman spiders in the Retrolateral Tibial Apophysis (RTA) clade is strongly supported (Moradmand et al., 2014, Ramírez, 2014, Wheeler et al., 2017, Fernández et al., 2018). Moradmand et al. (2014) inferred the Sparassidae as sister to the rest of the RTA clade but did not propose placement of the family beyond this. Wheeler et al. (2017) recovered sparassids as sister to the Oval Calamistrum clade + Dionycha, but this support was weak, and the position of Sparassidae within the RTA clade remained unresolved. Garrison et al. (2016) did not include sparassids, despite including many other major members of the RTA clade. The largest phylogenomic analysis of spiders to date placed the clade con- taining Sparassidae and the marronoid clade (sensu Wheeler et al., 2017) sister to the rest of the RTA clade (Fernández et al., 2018). Azevedo et al. (2022) also recovered sparassids as sister to the marronoid clade. Nearly a third of sparassid genera are not assigned to a formally described subfamily (Table 1). Currently eleven subfamilies have been described: Chrosiodermatinae Simon, Clastinae Simon, Deleninae Hogg, Eusparassinae Järvi, Heteropodinae Thorell, Palystinae Simon, Poly- betinae Järvi, Sparassinae Bertkau, Sparianthinae Simon, Staianinae Simon, and Tibellomatinae Simon. Four previous studies have published phylogenetic hypotheses to evaluate internal relationships within the Sparassidae (Agnarsson & Rayor, 2013, Moradmand et al., 2014, Wheeler et al., 2017, Tong et al., 2019). Furthermore, some genera are either known or suggested to be paraphyletic (Agnarsson & Rayor, 2013, Moradmand et al., 2014). Based on morphology, Hirst (1990) split the delenine genus Isopeda (I.) L. Koch 1875, into the genera Isopeda, Holconia Thorell 1877, and Isopedella (Iso.) Hirst 1990. These relationships are not supported in phylogenetic analyses, with all three genera rendered paraphyletic, despite forming a clade when examined as a whole (Agnarsson & Rayor, 2013). The genus Olios Walckenaer 1837, formerly the largest genus of sparassids, was until recently known to be paraphyletic but was reduced by Jäger (2020a) to 87 species distributed in Africa, Southern Europe, and Asia. Rheims and collaborators (Rheims, 2007a; Rheims & Jäger, 2008, 2022; Rheims, 2010a, 2010c, 2015, 2019a) have moved many of the New World “Olios” species into other previously known or new genera, such as Vindullus Simon 1880a, Macrinus Simon 1887, Caayguara Rheims 2010a, Curicaberis Rheims 2015, among others. While the majority of species are solitary hunters beyond a short period of transient maternal care of egg sacs and newly emerged young, a few sparassids have evolved sociality. To date, five species (Delena cancerides Walckenaer 1837, Delena lapidicola (Hirst, 1991a), Delena melanochelis (Strand 1913), Delena spenceri (Hogg, 1903), and an un- identified Damastes sp. Simon 1880a have been observed to live in long lasting mother-offspring groups (Agnarsson & Rayor, 2013, Yip & Rayor, 2014, Rayor unpublished data). These social species are char- acterized as ‘prolonged subsocial’ and consist of colonies of an interac- tive family group with a single adult female and her immature or newly mature offspring, who remain together in a retreat to have a ‘prolonged’ association lasting approximately five months to a year depending on the species (Yip & Rayor, 2014). Until the discovery of prolonged sub- sociality in a Damastes sp. from Madagascar, all other social sparassids were in the endemic Australian Deleninae, necessitating further phylo- genetic investigation to understand the comparative social evolution of the group. Fig. 1. Survey of some of the adult female huntsman spiders in this study. A: Pandercetes cf. malleator. B: Pandercetes cf. nigrogularis on plastered egg sac embedded into curl of leaf. C: Barylestis occidentalis with plastered egg sac and first instar young. The young have emerged from a split between the front and back sheets of the egg sac. D: Heteropoda jugulans with plastered egg sac. E: Heteropoda lunula immature female. F: Heteropoda venatoria with lenticular egg sac carried under abdomen. G: Heteropoda boiei. H: Damastes sp. gravid. I: Delena cancerides with plastered egg sac, non-feeding first instar young. J: Delena gloriosa with plastered egg sac. K: Neosparassus calligaster with round egg sac, silken cage, first instar. White at base of silken cage is a bottle cap pulled into the silk. K: Beregama aurea on lenticular to almost round egg sac depending on egg mass, silk attaching sac to substrate is clear. L: Pediana horni. M: Isopeda villosa. N: Isopeda leishmanni on lenticular egg sac with a newly opened emergence hole. All Deleninae females chew a hole in the egg sac for the young to emerge from. O: Isopedella pessleri with first instar young on lenticular egg sac. P: Isopeda canberrana within an hour after young emerge from the chewed hole in sac. The young emerge as nymphs and upon emergence molt to first instar with longer legs. Silk lines attach the egg sac to the bark. Q: Isopedella leai in silk cage with access hole on bottom. R: Isopedella conspersa outside a silk cage with a lenticular egg sac. S: Holconia nigrigularis with newly laid lenticular egg sac with silk attachments. T: Holconia insignis on lenticular egg sac with silk at- tachments. Photos of Beregama aurea (L) and Iso. conspersa (S) by Alan Henderson, all other photos by Linda Rayor. J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 3 Table 1 Information on taxonomic placement of 91 current genera in sparassid subfamilies. Genera with molecular data represented in this study are bolded. Subfamily Genera Included Status Distribution Notes Chrosiodermatinae Chrosioderma Simon 1897a Monotypic Africa (Madagascar) Clastinae Clastes Walckenaer 1837 Monotypic Oceania Deleninae Delena Walckenaer 1837, Beregama Hirst 1990, Holconia Thorell 1877, Isopeda L. Koch 1875, Isopedella Hirst 1990, Pediana Simon 1880a, Neosparassus Hogg 1903, Typostola Simon 1897a, Zachria L. Koch 1875, some former Olios rendered genus incertae sedis Monophyletic (Agnarsson & Rayor 2013, Wheeler et al., 2017, Moradmand et al., 2014) Oceania See Jäger (2020a) for specifics on subset of former Olios now designated in Deleninae incertae sedis Eusparassinae Arandisa Lawrence 1938, Carparachne Lawrence 1962, Eusparassus Simon 1903a, Leucorchestris Lawrence 1962, Microrchestris Lawrence 1962, Orchestrella Lawrence 1965, Palystella Lawrence 1928, Pseudomicrommata Järvi 1912 Paraphyletic: subset not related to Eusparassus forms monophyletic African clade (Moradmand et al., 2014); Eusparassus monophyletic Africa, Asia, Europe, South America Heteropodinae (sensu stricto) Barylestis Simon 1909, Bhutaniella Jäger 2000, Heteropoda Latreille 1804, Martensikara Jäger 2021, Martensopoda Jäger 2006, Pandercetes L. Koch 1875, Platnickopoda Jäger 2020c, Pseudopoda Jäger 2000, Sinopoda Jäger 1999b, Spariolenus Simon 1880a, Thunberga Jäger 2020b, Yiinthi Davies 1994 Monophyletic (Agnarsson & Rayor 2013, Moradmand et al., 2014) Africa, Asia, Oceania; Barylestis variatus introduced widely in Europe; Heteropoda venatoria introduced nearly worldwide Heteropodinae (sensu lato) As above, and including: Anaptomecus Simon 1903b, Guadana Rheims 2010d, Sparianthina Banks 1929, some former Olios rendered genus incertae sedis Polyphyletic, with Meri cf. sanctivincenti included the sole representative in Moradmand et al. (2014), but see text North America, South America Caayguara moved to incertae sedis by Pinto & Rheims (2016); see Jäger (2020a) for specifics on subset of former Olios now designated incertae sedis Palystinae Anchonastus Simon 1898b, Palystes L. Koch 1875, Panaretella Lawrence 1937, Parapalystes Croeser 1996, Sarotesius Pocock 1898, at least one former Olios rendered genus incertae sedis Monophyletic (Agnarsson & Rayor 2013, Moradmand et al., 2014) Africa, Asia See Jäger (2020a) for specifics on subset of former Olios now designated incertae sedis Polybetinae Polybetes Simon 1897a Monotypic South America Sparassinae Adcatomus Karsch 1880, Cebrennus Simon 1880b, Cerbalus Simon 1897a, Curicaberis Rheims 2015, Olios Walckenaer 1837, Macrinus Simon 1887, Micrommata Latreille 1804, Quemedice Mello-Leitão 1942, Sivalicus Dyal 1957, Vindullus Simon 1880a, some former Olios rendered genus incertae sedis Polyphyletic (Moradmand et al., 2014) Asia, Africa, Europe, North America, South America Rheims (2015) placed Curicaberis in Sparassinae; see Rheims (2007b) and Jäger (2020a) for specifics on subset of former Olios now designated incertae sedis Sparianthinae Decaphora Franganillo 1931, Diminutella Rheims & Alayón 2018, Extraordinarius Rheims 2019b, Neostasina Rheims and Alayón 2016, Pleorotus Simon 1898c, Rhacocnemis Simon 1898b, Sparianthis Simon 1880a, Stasina Simon 1877, Stipax Simon 1898c, Thelcticopis Karsch 1884, Thomasettia Hirst 1911, Uaiuara Rheims 2013 Sister to the rest of Sparassidae ( Moradmand et al., 2014, Wheeler et al., 2017); Sparianthinae represented only by Neostasina in Tong et al. (2019), so monophyly not evaluated Asia, Africa, North America, Oceania, South America Diminutella included by Rheims & Alayón (2018), Extraordinarius included by Rheims (2019), Neostasina included by Rheims & Alayón (2016), Uaiuara placed here by Rheims (2013) Staianinae Staianus Simon 1889 Monotypic Africa (Madagascar) Tibellomatinae Tibellomma Simon 1903a Monotypic South America (Venezuela) Described only from immature, see text Generic group 1 sensu Moradmand et al., 2014 Damastes Simon 1880a, Megaloremmius Simon 1903a Designated by Moradmand et al., 2014 Africa (primarily Madagascar) Generic group 2 sensu Moradmand et al., 2014 Gnathopalystes Rainbow 1899, Prychia L. Koch 1875, Tychicus Simon 1880a Designated by Moradmand et al., 2014 Asia, Oceania Generic group 3 sensu Moradmand et al., 2014 Remmius Simon 1897b, Rhitymna Simon 1897b Designated by Moradmand et al., 2014 Africa, Asia “African Clade” sensu Moradmand et al., 2014 Arandisa, Carparachne, Leucorchestris, May Jäger & Krehenwinkel 2015, Microrchestris, Palystella, Pseudomicrommata Designated by Moradmand et al., 2014 Africa May included by Jäger & Krehenwinkel (2015) Arandisa, Carparachne, Leucorchestris, Microrchestris, Palystella, Pseudomicrommata all still formally in Eusparassinae incertae sedis Berlandia Lessert 1921, Caayguara Rheims 2010a, Deelemanikara See note at Heteropodinae s.l. regarding inclusion of Caayguara; (continued on next page) J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 4 Based on long-term study by LSR, most huntsman species reach adulthood in their tenth instar, with individual Delena lapidicola starting to disperse in the fifth or sixth instar or later; D. melanochelis, D. spenceri, and Damastes sp. in the sixth instar or later; and D. cancerides occa- sionally in the eighth instar, but most as penultimate ninth instars or young adults. Prolonged subsocial species are characterized by a single breeding adult female and the simultaneous presence of multiple clutches of her offspring of all ages in the same retreat, sharing prey, and extensive interaction, tolerance, and (in D. cancerides) intolerance to older immigrants (Rowell & Avilés, 1995, Auletta & Rayor, 2011, Yip et al., 2012, Yip & Rayor 2013, Yip & Rayor, 2014, Jones & Rayor, 2017). Subadult or recently mature females disperse independently to establish their own retreats, and similarly aged males disperse to find receptive females. In all species studied to date, a single adult female establishes a colony and other mature females are not tolerated. Addi- tionally, all five prolonged subsocial species have delayed development where the spiderlings emerge from the egg sac at the first instar with large yolk-filled abdomens, but do not feed until they molt to the second instar (Cardillo & Rayor, in preparation, using terminology which refers to the first instar to emerge from the egg sac as the first instar or one instar earlier than referred to in earlier Rayor publications). All other solitary and subsocial species start foraging during the first instar. Like other sparassids, but unlike most subsocial and social spiders in other families, prolonged subsocial species do not live on prey capture webs, do not exhibit a skewed sex ratio, and do not show evidence of the inbreeding typical of cooperative ‘social’ spiders (Avilés & Guevara, 2017). Instead, they are found in bark or rock retreats. Most other social spiders use webs as a substrate for chemical and vibrational cues, so the presence of social behavior in a group lacking such a hallmark of spider communication is especially noteworthy (Avilés, 1997, Avilés & Gue- vara, 2017). Two more delenine species exhibit subsocial behavior in the labo- ratory: Isopedella leai (Hogg, 1903) and I. pessleri (Thorell, 1870). During the four to five weeks (late third instar) that these two species remain with their mother and siblings, they are notable for the relatively high frequency of prey sharing, and tolerant associations compared to the solitary huntsman species (Rayor & Giulian unpublished data, Yip & Rayor, 2014). Solitary species typically disperse during the second instar. With varied life history, behavioral, physiological, and develop- mental traits, sparassids represent a model clade (sensu Sanger & Raja- kumar, 2019). This diversity makes phylogenetic study especially informative for understanding the evolution of social behavior. We infer a robust molecular phylogeny of the Sparassidae by broadening taxon sampling in the group. We then employ this phylogeny to evaluate hy- potheses about life history traits and social evolution based on extensive field and laboratory observations. Group living in huntsman spiders is characterized by mothers who remain associated with one or more co- horts of their offspring for long periods. Thus, we hypothesized that prolonged subsociality could be phylogenetically associated with certain life history traits, such as more permanent or well-protected retreats, less movement while guarding eggs, spatial or developmental charac- teristics that may facililtate interactions among group-members, or even a longer lifespan. In contrast, we anticipated that solitary species should have less permanent or protective retreats, could be more vagile while guarding egg sacs or have shorter lifespans. As such, we tested life his- tory traits, including retreat features, egg sac structure and attachment, development, and lifespan, across the Sparassidae to determine whether certain character states are ssociated with group-living in a phylogenetic context. 2. Materials and methods Life history data for 40 species were based upon laboratory and field observations by L. Rayor and were collected between 2002 and 2021. These data include observations on mother-offspring dynamics and so- ciality, egg sac structure, how the egg sac was attached to the retreat or carried, retreat type, modifications to the retreat, adult female body mass and cephalothorax width, age at sexual maturity, and lifespan for the following subfamilies: Sparianthinae (represented by Thelcticopis), Heteropodinae, Palystinae, Sparassinae (represented by a true Olios and one “Olios”), and Deleninae. Legs, or whole bodies, in the case of spi- derlings, were pulled from representative specimens for molecular analysis and stored in 95% ethanol in a − 20 ◦C freezer until extraction. Additionally, voucher specimens representing species in the molecular and life history dataset were deposited at the National Museum of Natural History (NMNH, USA). A total of 82 DNA samples were extracted in this study. PCR was performed for nuclear genes H3 and 28S rRNA and mitochondrial genes 16S rRNA and COI using primers described in Palumbi et al. (1991), Folmer et al. (1994), Colgan et al. (1998), Hedin & Maddison (2001), and Vink et al. (2005). Protocols were adapted from Agnarsson et al. (2007, 2013b) and Agnarsson (2012). After purification with ExoSAP-IT (Thermo Fisher, Waltham, MA, USA), samples were sent to the Cornell Genomics Facility (Ithaca, NY, USA) for bidirectional Sanger sequencing. Sequences were assembled using Geneious Prime 2020.0.5 (https://www.geneious.com/). Sequences generated in this study were combined with sequences from 201 additional samples from GenBank to increase taxon representation within the Sparassidae and to provide outgroups. Outgroups included the following: Deinopis spinosa Marx 1889 (Deinopidae), Uloborus diversus Marx 1898 (in Banks 1898; (Ulo- boridae)), Oecobius Blackwall 1862 (Oecobiidae), Uroctea durandi ((Latreille, 1809); Oecobiidae), Peucetia viridans ((Hentz, 1832); Oxy- opidae), Dolomedes tenebrosus Hentz 1844 (Pisauridae), Salticus scenicus (Clerck, 1757; Salticidae), Selenops muehlmannorum Jäger & Prax- aysombath, 2011 (Selenopidae), and Tibellus chamberlini Gertsch 1933 (Philodromidae). Sequences were aligned using MAFFT and were manually examined using Mesquite version 3.6 (Katoh & Standley, 2013, Maddison & Maddison, 2018). Aligned sequences were concate- nated and phylogenies were inferenced in IQ-TREE using ModelFinder and ultrafast bootstraps (UFBoot) as support values (Nguyen et al., 2015, Kalyaanamoorthy et al., 2017, Hoang et al., 2018). With the IQ-TREE Table 1 (continued ) Subfamily Genera Included Status Distribution Notes Jäger 2021, Defectrix Petrunkevitch 1925, Dermochrosia Mello-Leitão 1940, Exopalystes Hogg 1914, Geminia Thorell 1897, Irileka Hirst 1998, Keilira Hirst 1989, Nolavia Kammerer 2006, Nungara Pinto & Rheims 2016, Meri Rheims & Jäger 2022, Origes Simon 1897b, Paenula Simon 1897b, Prusias O. Pickard- Cambridge 1892, Sadala Simon 1880, Sagellula Strand 1942, Stasinoides Berland 1922, Strandiellum Kolosváry 1934, some former Olios rendered genus incertae sedis Africa, South America, North America, Asia, Oceania Nungara included by Pinto & Rheims (2016); see Jäger (2020a) for specifics on subset of former Olios now designated incertae sedis J.A. Gorneau et al. https://www.geneious.com/ Molecular Phylogenetics and Evolution 174 (2022) 107530 5 phylogeny as a starting tree, a maximum likelihood phylogeny was also inferenced using the program RAxML (Stamatakis, 2014). Due to a lack of convergence, a starting tree was used for Bayesian inference in MrBayes (Huelsenbeck & Ronquist, 2001, Ronquist & Huelsenbeck, 2003). Divergence dating was implemented in BEAST, calibrated with fossil outgroups, while a fossil sparassid exists, it is not useful for cali- bration purposes (Drummond et al., 2012, Suchard et al., 2018, Bouckaert et al., 2019, Magalhães et al., 2020). Models of molecular evolution were selected using jModelTest2 (Darriba et al., 2012). Fossil outgroups included Zamilia aculeopectens Wunderlich 2015 (Oecobiidae) for the node representing Oecobiidae, Oxyopes succini Petrunkevitch 1958 (Oxyopidae) for Oxyopidae + Pisauridae, Almolinus ligula Wun- derlich, 2004 (Salticidae) for the node containing the outgroups Salt- icidae + Philodromidae, and ‘Selenops’ sp. indet. Wunderlich, 1988 (Selenopidae) for the node with outgroups Selenopidae sister to Salt- icidae + Philodromidae BEAST runs were examined using the program Tracer (Rambaut et al., 2018). Trees were initially examined using FigTree (Rambaut, 2018). MAFFT, MrBayes, jModelTest2, and BEAST were all run using the CIPRES Science Gateway (Miller et al., 2010). Stochastic character mapping was selected as the method of choice for trait mapping to allow for examination of character evolution among nodes and the generation of posterior probabilities from stochastic character map runs at each node. This was completed in the R using the packages corHMM, phylotools, and phytools (Revell, 2012, Beaulieu et al., 2013, Zhang et al., 2017, R Core Team, 2022). Using stochastic character maps also enabled a pairwise examination of correlation be- tween life history traits and sociality through the D-test, developed by Huelsenbeck et al. (2003) for identifying correlations of morphological characters and implemented in phytools. Morphological data for the subfamily Deleninae as well as the social species of Damastes, currently undescribed, were scored by C.A. Rheims (see the corresponding Data in Brief paper by Gorneau et al. (in review) for more information as well as images of the Damastes species studied). These characters were mapped on the tips of the Deleninae + Damastes phylogeny using the packages phylotools, phytools, and RColorBrewer (Revell, 2012; Neuwirth, 2014; Zhang et al., 2017). For more detailed information on all materials and methods with respect to data collection and analysis including specific model param- eters for phylogenetic inferences, please refer to the corresponding Data in Brief article (Gorneau et al., in review). 3. Results 3.1. Sequencing results and matrix completeness Of 82 samples, 70 had at least two genes that sequenced successfully and were included in these analyses. The concatenated matrix with GenBank data included was a total of 2173 basepairs in length (GenBank data sourced from: Agnarsson & Blackledge, 2009, Blackledge et al., 2009, Crews & Gillespie, 2010, Agnarsson & Rayor, 2013, Moradmand et al., 2014, Wheeler et al., 2017, Tong et al., 2019). Within this matrix were 1687 distinct patterns, 1142 parsimony-informative sites, 303 singleton sites, and 728 constant sites. For H3, 12 of 271 samples lacked sequence data. For 28S rRNA, 27 of 271 samples lacked sequence data. For COI, 38 of 271 samples lacked sequence data. For 16S rRNA, 21 of 271 samples lacked sequence data. Overall, the entire matrix contained about 14% missing data. 3.2. IQ-TREE maximum likelihood phylogeny of Sparassidae The IQ-TREE maximum likelihood inference included a total of nine outgroup taxa and 262 ingroup samples, representing 37 of the 89 described genera. A topology in support of a monophyletic Sparassidae was recovered (Fig. 2, UFBoot = 100). Support for the monophyly of subfamilies in the Sparassidae was varied, consistent with previous work (Agnarsson & Rayor, 2013, Moradmand et al., 2014). The sister group to all other huntsman spiders, the Sparianthinae, were recovered as monophyletic (UFBoot = 96). The Heteropodinae sensu stricto were recovered as monophyletic (UFBoot = 98), and while the Heteropodinae sensu lato (represented with the inclusion of Sparianthina Banks, 1929) were also recovered as monophyletic, the support was comparably low (UFBoot = 78). Moradmand et al. (2014) considered Meri cf. sancti- vincenti Simon 1898a as part of Heteropodinae sensu lato, though this representative is not considered part of the group in this study, as its inclusion renders the group paraphyletic. A clade of exclusively Neotropical sparassids formerly placed in Olios was recovered as monophyletic (UFBoot = 84) and weakly recovered as sister to the Neotropical Polybetinae, represented solely by Polybetes pythagoricus (Holmberg, 1875), and thus unable to be evaluated on itself. The Paly- stinae were recovered as monophyletic (UFBoot = 100). The monotypic Staianinae, represented by a single Staianus Simon 1889, was recovered as sister to two undetermined “Olios” from the pet trade (UFBoot = 96). The genus Damastes was recovered as monophyletic (UFBoot = 100), but weakly supported nodes make it difficult to make any confident in- ferences about which group it is most closely related to. Micrommata virescens (Clerck, 1757), the type of the subfamily Sparassinae, was recovered with poor support sister to Eusparassinae + Deleninae, (UFBoot = 67). The remaining members of the subfamily examined (Olios, Cerbalus Simon 1897a) formed a monophyletic group with “Olios” giganteus Keyserling 1884 and related taxa from the Neotropics (UFBoot = 97), rendering the Sparassinae non-monophyletic. The Eusparassinae were recovered as paraphyletic, with the proposed members of the ‘African clade” sensu Moradmand et al. (2014) recovered as their own group and Eusparassus Simon 1903a recovered as mono- phyletic (UFBoot = 100), and weakly recovered as sister to the Dele- ninae (UFBoot = 57). The endemic Australian Deleninae were recovered as monophyletic (UFBoot = 67). An uncollapsed view of the tree is available in Fig. S1. The results of a RAxML inference, as well as a Bayesian inference via MrBayes, both using the IQ-TREE phylogeny as a starting tree, were largely similar and are available in the corresponding data paper (Gorneau et al., in review). 3.3. BEAST fossil-calibrated estimated divergence of Sparassidae The results of the BEAST fossil-calibrated divergence time for the Sparassidae suggest an estimated stem age of ~ 98 million years and a crown age of ~ 90 million years (Fig. 3). The crown age for the social genus Delena Walckenaer 1837 is approximately 24 million years old, representing the oldest generic lineage within the Deleninae. The crown age for the Sparianthinae, sister to all other huntsmen, represented an age of approximately 79 million years old. The 95% confidence intervals overlapped for most inferences. 3.4. Stochastic character mapping and D-test A solitary lifestyle with transient parental care limited to maternal guarding of the egg sac and newly emerged young is the ancestral state for sparassids. The ancestral state reconstruction supports four inde- pendent origins of social behavior, with two origins of prolonged sub- sociality, one in Delena and one in Damastes, and two origins of subsociality in Isopedella (Fig. 4). The final two origins of sociality are in Isopedella, with Isopedella leai and Isopedella pessleri as the only huntsman species with a brief period of subsocial behavior, but not prolonged subsocial behavior. The ancestral state for the subfamily Deleninae in- dicates solitary origins, except in the genus Delena. The ancestral state of social behavior in the genus Delena is prolonged subsocial with one reversal to solitary behavior in Delena gloriosa (Rainbow, 1917). With only one representative for Damastes, an ancestral condition could not be inferred for the genus. The ancestral egg sac structure for the Sparassidae excluding Thelc- ticopis Karsch 1884 (Sparianthinae) is inferred as plastered (Fig. 4, see Fig. 1B, C, D, I, and J). Lenticular egg sacs are the ancestral state for the J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 6 Fig. 2. Maximum likelihood phylogeny of the huntsman spiders with UFBoot support values inferred using IQ-Tree. Triangles represent collapsed nodes of multiple specimens representing a single taxon and the length corresponds to the branch length range for those collapsed specimens. Sample size is indicated in parentheses for these nodes. Colors correspond to different subfamilies. Scale of 0.08 refers to branch lengths. J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 7 Deleninae (see Fig. 1F, L, O, P, Q, S, T, and U). However, plastered egg sacs are the ancestral condition for the social genus Delena and in the Heteropodinae. The genus Damastes also has a plastered egg sac type, but an ancestral condition could not be inferred for the genus with just one representative. While plastered egg sacs are ancestral to the Het- eropodinae, including the genera Pandercetes L. Koch 1875, Barylestis Simon 1909, and Heteropoda Latreille 1804, lenticular egg sacs are ancestral to the genus Heteropoda excluding Heteropoda jugulans (L. Koch 1876). Spherical egg sacs are characteristic of Olios, Neosparassus Hogg 1903, and Thelcticopis (though note with only one representative for the latter two, an ancestral state could not be inferred for these genera; see Fig. 1K). The ancestral state for egg sacs is ‘attached by silk lines’ in the Sparassidae, and in Sparassidae sister to Thelcticopis, ‘attached to sub- strate’ (Fig. 4). For the clade containing Olios + Eusparassus +Deleninae, the ancestral state is an egg sac attached by silk lines (see Fig. 1L, T, Q, U). In the Heteropodinae, the ancestral state for egg sac support is an immobile egg sac attached to the substrate, but a clade in the speciose genus Heteropoda evolved so that the lenticular egg sac that is con- structed on the substrate is subsequently pulled off and carried freely under the mother’s venter to allow mobility while guarding the egg sac (Fig. 1F). Only two species in this study have egg sacs bound in leaves Fig. 3. Divergence date estimates from BEAST maximum clade credibility inference. Circles at each node correspond to posterior probabilities as indicated by color scale to the upper left of the figure. The number at each node corresponds to the age of the clade in millions of years, and the light blue rectangles represent the 95% highest posterior density (HPD). Scale of 20.0 refers to millions of years. Geologic time scale at bottom of figure, with periods, epochs, and subdivisions created using R packages phytools, phyloch, strap, and coda (Plummer et al., 2006, Heibl 2008, Revell 2012, Bell and Lloyd 2015). “Q” referring to Quaternary Period. The illustration is of Heteropoda boiei. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 8 and attached by silk: Palystes superciliosus L. Koch 1875 and Beregama cordata (L. Koch 1875). Except for Thelcticopis and Heteropodinae, the ancestral state for retreat location is under bark for all Sparassidae included in this study. The ancestral state for retreat location in the Heteropodinae is ambig- uous, though the clade containing Barylestis and Pandercetes has an open retreat (Fig. 4). The ancestral state for retreats is the large, diverse genus Heteropoda is ambiguous. The ancestral condition for retreat modifications for all Sparassidae, excluding Thelcticopis and the Heteropodinae, is silk bonded to the substrate (Fig. 4). In the Deleninae, all members have at least some sort of retreat modification, the ancestral condition for retreat modifications being silk bonded to substrate. Retreats modified to include silken cages are found in Neosparassus sp., Isopedella conspersa (L. Koch 1875; Dele- ninae), Iso. leai, Thelcticopis, Olios, and Eusparassus (Fig. 1K, R, S). Moradmand & Jäger (2012) refer to this retreat type as a “papery retreat” in Eusparassus. The lack of retreat modifications is ancestral and highly conserved within the Heteropodinae. Approximate adult female body mass and adult female cephalo- thorax width serve as proxies for overall body size. Large variation in body size occurs throughout the sparassids, with no clear ancestral states for most major lineages and no apparent evolutionary trends associated with body size. There is little variation in age of sexual maturity and life span among the sparassids. Most sparassid females reach sexual matu- rity at about 1 year and live to approximately 2.5 years. Smaller species reached sexual maturity somewhat earlier and died closer to 1.5 to 2 years. The only notable exceptions were three very large species: Bere- gama aurea (L. Koch 1875) mature at 1.7 years and commonly live to over 3.5 years old, Damastes sp. which matures at 17 + months and lives over 3.3 years, and a sole Typostola barbata (L. Koch 1875) that lived to four years. D-tests explored the relationship between patterns of group-living and life history traits. Starting to eat in the first instar out of the egg sac was significantly corrected with a solitary lifestyle (p = 0.006), while delaying eating until the second instar was significantly associated with prolonged subsociality (p = 0.005). Lenticular egg sacs were associated with a solitary lifestyle (p = 0.029), while plastered egg sacs were correlated with prolonged subsociality (p = 0.02). Immobile egg sacs attached to the retreat by silk lines were associated with being solitary (p = 0.026), while immobile egg sacs plastered to the retreat were correlated with prolonged subsociality (p = 0.013). A retreat under bark was correlated with prolonged subsociality (p = 0.035). Silk bonding the substrate (whether bark, rock, or leaf) together to form a more secure, enclosed retreat is correlated with prolonged subsociality (p = 0.039). In Fig. 4. Phylogenetic patterns of life history traits in sparassids based on stochastic character mapping for the following characters: level of sociality (square), egg sac structure (pentagon), how egg sac is attached or carried (triangle), retreat location (diamond), and retreat modification (star). The colors correspond to different character states. All red squares represent both prolonged subsociality and delayed development. All other squares represent solitary or subsocial species which have non-delayed development. An all-black shape indicates no majority ancestral state for that node. A black outline indicates an ancestral state with ≥ 75% posterior probability, while no black outline indicates ≥ 50% posterior probability of ancestral state. Along the tree, downstream ancestral nodes are the same unless otherwise indicated. Exceptions to the ancestral nodes at the tips are indicated with a corresponding icon next to the tip label. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 9 terms of retreat size relative to body size, a lack of a retreat was asso- ciated with solitary behavior (p = 0.015), and medium to large retreats were associated with prolonged subsociality (p = 0.011 and p = 0.001, respectively). Solitary females with eggs or young were associated with small retreats (p = 0.007), prolonged subsocial females with eggs or young were associated with larger retreats (p = 0.06 for medium retreats and p = 0.029 for large retreats). No life history traits were observed to be correlated specifically with the subsociality observed in Isopedella leai and Isopedella pessleri, nor was there a correlation between body size and level of sociality. 3.5. Mapping of deleninae and Damastes morphology In the Deleninae, morphological features including eye patterns, and male and female sexual anatomy generally track phylogeny. Delena are clearly separated from the remaining genera by the presence of pitted fovea rather than a longitudinal fovea, the lack of spermathecal sacs in the female vulva, and posterior median eyes closer to each other than to the posterior lateral eyes (Fig. 5). Prolonged subsocial Damastes are most similar morphologically to the Delena, with seven of the eleven traits similar and differences primarily in details of the eye patterns and the Fig. 5. Cladogram of subfamily Del- eninae and Damastes with morpho- logical traits assessed by C.A. Rheims represented to the right. Different colors indicate a different character state, and question mark indicates a degree of uncertainty to the character state for that species. The last six characters correspond to male (four characters) and female genitalia characters (two characters), respec- tively. Illustrations by C.A. Rheims are representative of the genitalic variation within the subfamily and with Damastes. Abbreviations for characters are as follows: prosoma (fovea, posterior eye row shape (PER), anterior median eye (AME) diameter, AME-AME and PME-PME interdistances), male palp (embolic sclerite (EmS), conductor sclerotized base (CSB), tegular apophysis (TA), flange (TF)) and female epigyne and vulva (epigynal sclerite (EpS), sper- mathecal sacs (SS)). Scale lines: 1 mm. Illustrations of other taxa, as well as a breakdown of characters and character states, can be found in the Supplementary Material of the corre- sponding Data in Brief paper (Gor- neau et al., In Review). J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 10 absence of an embolic sclerite in the female epigyne, though this is not expected to be a direct correlate or cause of sociality in these genera. The clade containing Zachria L. Koch 1875, Neosparassus, Beregama Hirst 1990, Typostola Simon 1897a, and Pediana Simon 1880a can be distinguished from Isopeda, Isopedella, and Holconia by the male palps lacking a sclerotized conductor base, a delenine tegular apophysis and a tegular flange and female epigyne lacking an epigynal sclerite (although these traits can be present in one or both Beregama). There are few morphological traits which are distinct within the confusing Isopeda- Isopedella-Holconia clade. Although all three genera have a tegular apophysis in the male palp, it is knob-shaped in Holconia and hood- shaped in Isopeda and Isopedella. There are no clear patterns that clearly distinguish Isopeda from Isopedella. The subsocial Isopedella leai and Iso. pessleri differ from the other Isopedella and Isopeda by the absence of spermathecal sacs and size of the AME. Of interest, the sole delenine which is regularly diurnally active, Pediana regina (L. Koch 1875), is the only delenine species with a procurved posterior eye row. 4. Discussion 4.1. Phylogenetic results and implications This study represents the most comprehensive taxon sampling for a phylogeny of sparassids to date, building on previous work (Agnarsson & Rayor, 2013, Moradmand et al., 2014, Wheeler et al., 2017, Tong et al., 2019), and is the first to explore trends of life history evolution in the family. The undisputed monophyly of Sparassidae has been demonstrated previously in a variety of phylogenetic and morphological contexts (Rheims, 2007a, Ramírez, 2014, Agnarsson & Rayor, 2013, Moradmand et al., 2014, Wheeler et al., 2017), and is reinforced here. While previous work included exemplars of the Sparianthinae, multiple genera were not included, making it impossible to evaluate the mono- phyly of the subfamily itself (Tong et al., 2019). Evaluating the mono- phyly of the Sparianthinae was important as this subfamily is sister to the rest of the Sparassidae, is quite divergent as evidenced by the BEAST analysis, and has unique characteristics not present in other sparassids, including canoe-shaped eye tapeta, smaller retromarginal teeth and a median apophysis in the male palps (Jäger, 1998, Ramírez, 2014). The Sparianthinae were represented in our phylogeny by Thelcticopis and Neostasina Rheims & Alayón, 2016, and in our ancestral state re- constructions by Thelcticopis (Fig. 2, Fig. 4). The differences in life his- tory and morphology in this subfamily compared to the rest of the Sparassidae as well as the ~ 20 my difference in crown ages between Sparianthinae and the Sparassidae (~80 my for Sparianthinae, ~98 my for Sparassidae Fig. 5) warrants future work. As in previous studies, the monophyly of Deleninae and Hetero- podinae are each supported (Agnarsson & Rayor, 2013, Moradmand et al., 2014, Wheeler et al., 2017). Sparassinae and Eusparassinae were recovered as not monophyletic, consistent with previous phylogenetic treatment of the group (Moradmand et al., 2014). The type genus of Eusparassinae, Eusparassus, arises as a monophyletic clade sister to Deleninae, while the remaining representatives of the subfamily (Ara- ndisa, Pseudomicrommata, Palystella, Leucorchestris and Carparachne) arise sister to a clade including Olios, Cerbalus and several unidentified Neotropical sparassids. Similarly, the type genus of Sparassinae, Micro- mmata Latreille 1804, arises far removed from the remaining Spar- assinae, here represented by the genera Olios and Cerbalus. Previous taxonomic issues with the generic classification of Olios are supported by this study. Olios, as previously defined was paraphyletic, and the work done by Jäger (2020a) rendered this genus as mono- phyletic, by restricting it to 87 species distributed in Africa, southern Europe and Asia. Our results corroborate this new definition. True Olios, here represented by O. argelasius (Walckenaer, 1806), O. rossettii (Leardi 1901) and O. lamarcki (Latreille, 1806) are recovered as monophyletic in a clade including an unidentified species from Singapore. “Olios” giganteus, considered misplaced in Olios by Rheims (2010b) and Jäger (2020a), arises within a large clade of Neotropical sparassids distributed in North America (USA) and the Antilles. On the other hand, the two “Olios” in the pet trade, although originating from Cameroon (within the distribution of Olios), arise sister to Malagasy Staianus and far removed from the true Olios, which may provide some taxonomic insight into these Cameroonian “Olios” species. This study clarified relationships within the Deleninae beyond Agnarsson and Rayor (2013). Based on both genetics and morphology the inclusion of Beregama cordata in our analysis demonstrated that B. aurea and B. cordata do not constitute a monophyletic Beregama. In this and other studies (Cardillo & Rayor in preparation), B. aurea is unique for having many physiological features that do not align closely with other solitary delenine sparassids and should be studied further. Additional specimens of Typostola sp. ‘tiger’ in this analysis were recovered with moderate support as sister to Typostola barbata, consis- tent with a previous maximum likelihood inference of the group by Agnarsson and Rayor (2013). However, a Bayesian inference by Agnarsson and Rayor (2013) was unable to recover Typostola sp. ‘tiger’ as sister to Typostola barbata. More life history data and morphological comparisons involving a greater sampling of taxa from the genus Typostola will be needed to place Typostola sp. ‘tiger’ conclusively in Typostola. Agnarsson and Rayor (2013) found Zachria flavicoma L. Koch 1875, the type species for Zachria, nested within Neosparassus, so addi- tional taxon sampling is necessary to establish the taxonomic and phylogenetic position of Zachria with relation to Neosparassus. Hirst (1990) split the genus Isopeda into Isopeda, Isopedella and Hol- conia. The phylogenetic relationships inferred here are consistent with Agnarsson and Rayor (2013), in that while the three form a clade, the three genera, as currently defined, are not monophyletic. Based on these results we could synonymize all three genera. However, Holconia be- comes monophyletic by removing Holconia nigrigularis Simon 1908 from the genus, and this is corroborated by a number of distinctive morpho- logical traits (e.g. large body size, dorsoventrally flattened carapace, bold black-white–grey coloration) that distinguish the genus from both Isopeda and Isopedella. But, maintaining Holconia as a valid genus also means sinking Isopedella into Isopeda (including at least, H. nigrigularis, Isopeda (I.) vasta (L. Koch 1867), Isopedella (Iso.) conspersa, Iso. tindalei Hirst 1993, Iso. pessleri, Iso. maculosa Hirst 1993, Iso. victorialis Hirst 1993, and Iso. leai) and proposing at least two new genera (one, including at least: I. villosa and I. montana Hogg 1903; and the other, including at least: I. leishmanni Hogg 1903, I. parnabyi Hirst 1992, Iso. cana (Simon 1908), and Iso. frenchi (Hogg, 1903)). Thus, at this point, we refrain from making any major taxonomical changes, since this goes beyond the scope of the current paper. We believe that the study of the complex Isopeda-Isopedella requires further molecular and morpholog- ical investigation, with broader species representation, to determine the limits of possible groups and genera as well as their relationship with Holconia. Some subfamilies lack specimens that have yielded successful DNA extractions, including genera from monotypic subfamilies: Clastes Walckenaer 1837, Chrosioderma Simon 1897a, and Tibellomma Simon 1903a. Consistent with Agnarsson and Rayor (2013) and Moradmand et al. (2014), we were also unable to successfully extract molecular data from older Clastes or from Chrosioderma specimens. As Tibellomatinae exists based on a single description from an immature specimen, and as the type is lost, it is currently impossible to confirm this suspected case of nomen dubium (Simon, 1898b, Jäger & Rheims, 2008). We anticipate some remaining uncertainties, particularly in the backbone of the sparassid tree of life, to be due in part to some of the alignment issues encountered with rRNA loci, first discussed by Mor- admand et al. (2014), and encountered here. This affected our ability to include multiple outgroups from the RTA clade as well as affecting some of our alignments of ingroup taxa. Future molecular work on the group will likely employ a genomic approach, which will lessen the impact of alignment issues with single genes on phylogenetic inferences. J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 11 4.2. BEAST fossil-calibrated estimated divergence of Sparassidae The taxonomy and phylogenetic utility of the few known sparassid fossils are disputed (Agnarsson & Rayor, 2013, Magalhães et al., 2020). For example, one species, Zachria desiderabilis Petrunkevitch 1950, a fossil species tentatively placed by Petrunkevitch (1950) in the extant Australian endemic genus Zachria was designated genus indeterminate by Wunderlich (2012). To estimate the origins of sparassids without fossil sparassids, Agnarsson and Rayor (2013) used base pair substitution rates, and focused on the Deleninae, inferring a median age of 23 million years for the subfamily. Moradmand et al. (2014) and Tong et al. (2019) employed the use of an Eusparassus crassipes (C.L. Koch & Berendt, 1854) fossil to calibrate their inferences, but this use was defined as prob- lematic by Magalhães et al. (2020) and credited as the reason for older divergence estimates. Consequently, Magalhães et al. (2020) proposed a median age of ~ 130 million years for the node leading to sparassids (Caayguara + marronoid clade) by using fossils elsewhere in the spider tree of life. The results of the BEAST fossil-calibrated divergence time in this study resulted in a considerably younger estimation than the age inferred by Magalhães et al. (2020), and instead demonstrated a crown age for the sparassids of about 90 million years. Divergence estimates made with BEAST resulted in low ESS values for some non-nuisance parameters that were not resolved after combining three independent chains of 100,000,000 generations and by using a starting tree, but it is not expected to greatly impact divergence estimates. Obtaining well-resolved, time-calibrated phylogenetic inferences is essential to evaluate the monophyly of described taxa, resolve taxo- nomic issues, and to better understand evolutionary trends within the huntsman spiders, particularly with respect to life history. Having a more well-calibrated divergence inference will assist in exploring any potential associations of life history evolution to relevant geological events. 4.3. Stochastic character mapping and D-test Our hypothesis that prolonged subsociality is associated with a suite of life history traits that better accommodate longer-term associations between adult females and multiple cohorts of offspring was largely supported, even though some of the individual traits are shared by solitary species (Fig. 4). Prolonged subocial species are found in rela- tively large, sturdy, well-protected and long-lasting retreats under bark or rocks, with silk modifications to better affix the retreat components and limit access to the retreat from potential predators. They provide space for multiple large cohorts of developing spiders to remain near their mother. Long tenure in the retreat is associated with egg sacs that are permanently plastered in place. Delayed development is associated a period in which relatively immobile first instar spiderlings remain in an interactive social situation (Cardillo and Rayor, in preparation). When they begin to feed in the second instar, social species are considerably larger than comparable second instar spiderlings of all other solitary or subsocial sparassid species studied. However, our predictions about an association of sociality with patterns of earlier sexual maturity, longer lifespan, or greater body size were not supported. Many solitary genera either do not use retreats or utilize more ephemeral retreats in leaves, or rely, in part, on a silken cage to help define the retreat space and protect the eggs (Fig. 4). Other groups, such as most Deleninae, use smaller, long-lasting retreats under loose bark that could contain large clutches of early instar spiderlings, but are not large enough to accommodate spiders to near-maturity. In all solitary species, individuals in the first instar were mobile foragers that dispersed rapidly by the second or third instars. In summary, the asso- ciated life history traits of the prolonged subsocial species are suitable for facilitating large interactive groups of growing spiderlings that remain in their natal retreat for many months, while those of the solitary species are not. Despite independent origins of prolonged subsociality, Delena and Damastes share many life history traits: all Delena and the Damastes in this study have delayed development with a non-feeding first instar, plastered egg sacs that adhere to the substrate and modify their retreat with short silk bonds to bind the bark or rocks together and limit access to the retreat. All prolonged subsocial retreats are extremely narrow crevices, except for D. melanochelis which live in long narrow tubes of rolled bark. The most dorsoventrally flattened and large-bodied pro- longed subsocial species, D. cancerides, D. lapidicola, and Damastes, are all found in retreats less than 1 cm deep. We propose that plastered egg sacs may be an adaptation to minimize space in these narrow retreats, and possibly offer a single face for the adult female to guard from po- tential colony invaders. The single solitary Delena species whose behavior has been studied, Delena gloriosa, shares most of these ancestral traits (except they feed in the first instar) and our study and Agnarsson and Rayor (2013) indicate that it has secondarily lost sociality. There are other members of the genus Delena that have not been investigated in a behavioral or molecular context which may have implications for future inferences of the social evolution of this genus. Except for the solitary Delena gloriosa, each time another Delena species was found in the wild by LSR they were unexpectedly discovered to live in prolonged subsocial matrilineal groups (D. lapidicola, D. melanochelis, D. spenceri), suggesting that more members of the genus may be prolonged subsocial. It also suggests that further basic behavioral, developmental, and molecular research into the genus is recommended, particularly in the little known and recently described species (Delena convexa Hirst 1991a; D. loftiensis (Hirst, 1991b), D. tasmaniensis (Hirst, 1991b), D. kosciuskoensis, Hirst 1991b). Additionally, with the type of D. craboides Walckenaer 1837 lost, the species represents a nomen dubium and may in fact be D. cancerides (see Simon, 1880a). The convergent life history similarities in Delena and Damastes provide a blueprint of characteristics that should be explored when examining other sparassids for prolonged subsocial behavior. However, there were not obvious life history traits that were characteristic of the two subsocial species, Isopedella leai and Iso. pessleri, that differed from those of solitary species. Unlike the extensive field observations in the prolonged subsocial Delena, these subsocial Iso- pedella species have not been extensively observed in the field, only in the laboratory, and the extent of their association with their mother and siblings may differ in the field. The paucity of field observations is due to the anthropophilic tendencies of both species which are more often collected in (or removed from) homes than from the field and the short ≤ five-week period of association making observations during the period of association less common. Plastered egg sacs are found in a few other solitary Heteropodinae; Heteropoda jugulans, H. lunula (Doleschall, 1857), H. dagmarae Jäger & Vedel, 2005 (not included in this phylogenetic analysis), Pandercetes, and Barylestis. The plastered egg sacs of Pandercetes and some Heteropoda (H. jugulans, H. dagmarae) species are like those built by Delena and Damastes with a flat sheet fully adhered to the substrate and an outer sheet that fits around the egg mass. All lenticular egg sacs are con- structed as a distinct disk that is initially lightly plastered to the sub- strate during egg laying and then pulled off to be attached with guy-lines of silk or carried by the female. Unlike the construction of the other species plastered sacs, the plastered egg sacs of Barylestis and H. lunula are constructed like the lenticular sacs but with more extensive silk around the edges of the disk that tightly affixes it to the substrate through development of the eggs. When the eggs hatch and the post- embryo ‘eggs with legs’ swell the sac, the sides of the sac split open so the young emerge through the fissure (Fig. 1C). In contrast, Delena and Damastes mothers actively chew a hole in the egg sac through which the young emerge. All of the delenine females studied chewed holes in the egg sac for the young to emerge from (Fig. 1O, 1Q). The plastered sacs of Pandercetes and Barylestis are often camouflaged on the exterior of trees or dead leaves, and the plastered sac likely blends more effectively into the background without a shadow than would a lenticular egg sac. Lenticular sacs are the ancestral state in a clade of Heteropoda, as well as the clade of Deleninae including Beregama, Pediana, Typostola, Holconia, J.A. Gorneau et al. Molecular Phylogenetics and Evolution 174 (2022) 107530 12 Isopeda, and Isopedella. Spherical sacs were represented by our sole ex- emplars of Olios, Thelcticopis, Neosparassus, and Eusparassus. While we lacked multiple exemplars for Olios and Neosparassus, generally Olios (including species now formerly Olios following Jäger, 2020a) and Neosparassus build silken cages (Fig. 1K, Rayor, personal observations; Hirst, personal communication). At least some species of Isopedella, including Iso. conspersa and Iso. leai, build silk cages (Fig. 1R, 1S). The egg sacs of Palystes L. Koch 1875 are unique in that the shape of the egg sac itself appears to vary from lenticular to spherical depending on the egg mass, and females incorporate leaves and other vegetation into the outer structure of the sac with silk irregularly pulled off the surface of the sac where silk lines attach to the substrate (Hamilton-Attwell & Dippenaar-Schoeman, 2020). Beregama aurea sacs also vary from lenticular to spherical with the egg mass (Fig. 1L). The ancestral state for retreats in the Deleninae is under bark or in bark rolls. The exceptions, D. lapidicola and D. spenceri, live under exfoliating rocks on granite headlands or dolerite boulders, respectively, in rocky habitats with no trees and only small shrubs that do not have retreat spaces under their bark. The genus Heteropoda represents about 15% of all described sparassids, which are found in a wide range of habitats with equally varied retreat use including the lack of a retreat, in leaves, under rocks, and under bark. 4.4. Final remarks To resolve the taxonomic issues presented in this study, more basic research and taxonomy are needed on huntsman spiders. It will be particularly useful to acquire recent specimens viable for DNA extrac- tion, as well as information about the life history and behavior of understudied lineages such as Clastes Walckenaer 1837 (Clastinae) and Chrosioderma Simon 1897a (Chrosiodermatinae). Clastes freycineti Walckenaer 1837 is especially of interest regarding both placement and life history as morphological inferences recover the group as sister to Deleninae, which may have implications for social evolution of the family (Rheims, 2007a, Rheims personal communication). Like the other prolonged subsocial species studied here, C. freycineti produces a plastered egg sac and has been observed in putative social groups with the mother sharing prey with older offspring (L. Higgins, personal communication). Future phylogenetic work should center on resolving higher relationships of the genera currently designated incertae sedis, which represent almost one-third of all described sparassid species. 5. Conclusions We provide the most synthetized inference of Sparassidae by increasing the depth of delenine sampling, including taxa from previ- ously published work on the family, and by contributing new sequence data on key taxa. This inference further confirms that extensive phylo- genetic work remains to be done to further elucidate relationships and resolve taxonomic issues within the family. Results of nearly two de- cades of field and lab study by LSR analyzed via ancestral state recon- struction have revealed life history traits that have evolved in association with social behavior. This work provides a basis to study more social and life history traits in the Sparassidae to further under- stand predictors of prolonged subsocial behavior in the family. CRediT authorship contribution statement Jacob A. Gorneau: Conceptualization, Data curation, Formal anal- ysis, Funding acquisition, Investigation, Methodology, Project admin- istration, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Cristina A. Rheims: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Writing – review & editing. Corrie S. Moreau: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Linda S. Rayor: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We are indebted to Ivan Magalhaes, Dave Rowell, Ingi Agnarsson, Manuela Oliveira Ramalho and an anonymous reviewer for advice on the project, manuscript, and/or methodology. With gratitude we thank David Hirst, Frank Somma, Alan Henderson, D. Rowell, Nicky Bay, Jo- seph Koh, Linden Higgins, and Charles Haddad for extensive biological information and the first three for live specimens. Melbourne Museum Bugs Alive! and Lachlan Manning, Australian Museum, contributed live specimens. We gratefully thank Cole Gilbert, D. Rowell, Eric Yip, Fran- cesca VanDenBerg, Jacob Hurst, David Hirst, Daiqin Li, and David Court for assistance in the field. Extensive institutional and intellectual sup- port was provided by both D. Rowell and the Australian National Uni- versity and D. Li, National University of Singapore. Liam Revell (University of Massachusetts Boston) provided input on use of the D-test in his phytools package to determine correlations between sociality and life history. Sabrina Celis (Cornell University) illustrated the egg sacs and retreats. Funding for JAG includes the Jane E. Brody Undergraduate Research Award, the Fredric N. Gabler ’93 Memorial Research Endow- ment, and the CALS Alumni Association Academic Enrichment Program. Additionally, this work was supported by an NSF IOS grant [1916995] to CSM; Monster Bug Wars, Women’s International Science Collaboration (AAAS/ NSF) and the President’s Council of Cornell Women (Affinito- Stewart Grant) to LSR; and FAPESP grants [11/18694-3 and 15/18982- 0] to CAR. Appendix A. Supplementary material Voucher_information.xlsx – Excel file with information on vouchers representing exemplars for the molecular data contributed in this study. Specimens deposited in the Smithsonian arachnology col- lections. New_data_generated_GenBank.xlsx – Excel file with infor- mation for all individuals contributed for the molecular component of data collection study, including locality and date information where known, and GenBank accession numbers. Red cells indicate no sequence data for that gene, and gray cells indicate sequence data but only from a single sequencing direction (forward or reverse). Location/souce and date information also provided, where known. GenBank_sequences. xlsx – Excel file with accession numbers for sequences downloaded from GenBank. Figure_S1_IQ_TREE_Phylogeny_Uncollapsed.pdf – Maximum likelihood phylogeny of the huntsman spiders with UFBoot support values inferred using IQ-Tree. Colors correspond to different subfamilies. Scale of 0.08 refers to branch lengths. Supplementary data to this article can be found online at https://doi.org/10.1016/j.ymp ev.2022.107530. References Agnarsson, I., Blackledge, T.A., 2009. Can a spider web be too sticky? Tensile mechanics constrains the evolution of capture spiral stickiness in orb-weaving spiders. J. Zool. 278, 134–140. Agnarsson, I., Rayor, L.S., 2013. A molecular phylogeny of the Australian huntsman spiders (Sparassidae, Deleninae): implications for taxonomy and social behaviour. Mol. Phylogenet. Evol. 69, 895–905. Agnarsson, I., Maddison, W.P., Avilés, L., 2007. The phylogeny of the social Anelosimus spiders (Araneae: Theridiidae) inferred from six molecular loci and morphology. Mol. Phylogenet. Evol. 43, 833–851. J.A. Gorneau et al. https://doi.org/10.1016/j.ympev.2022.107530 https://doi.org/10.1016/j.ympev.2022.107530 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0005 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0005 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0005 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0010 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0010 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0010 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0015 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0015 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0015 Molecular Phylogenetics and Evolution 174 (2022) 107530 13 Agnarsson, I., Coddington, J.A., Kuntner, M., 2013a. Systematics: progress in the study of spider diversity and evolution. In: Spider Research in the 21st Century: Trends and Perspectives. Agnarsson, I., Gregoric, M., Blackledge, T.A., Kuntner, M., 2013b. Phylogenetic placement of Psechridae and the convergent origin of orb-like spider webs. J. Zool. Syst. Evol. Res. 51, 100–106. Agnarsson, I., 2012. Systematics of new subsocial and solitary Australasian Anelosimus species (Araneae: Theridiidae). Invertebr. Syst. 26, 1–16. Auletta, A., Rayor, L.S., 2011. Preferential prey sharing among kin not found in the social huntsman spider, Delena cancerides (Araneae: Sparassidae). J. Arachnol. 39, 258–263. Avilés, L., 1997. 23: Causes and consequences of cooperation and permanent-sociality in spiders. The evolution of social behavior in insects and arachnids 476–498. Avilés, L., Guevara, J., 2017. Sociality in spiders. Compar. Soc. Evol. 188–223. Azevedo, G.H., Bougie, T., Carboni, M., Hedin, M., Ramírez, M.J., 2022. Combining genomic, phenotypic and Sanger sequencing data to elucidate the phylogeny of the two-clawed spiders (Dionycha). Mol. Phylogenet. Evol. 166, 107327. Banks, N., 1898. Arachnida from Baja California and other parts of Mexico. P. Cal. Acad. Sci. 3 (1), 205–309. Banks, N., 1929. Spiders from Panama. B. Mus. Comparat. Zool. 69, 53–96. Beaulieu, J.M., O’Meara, B.C., Donoghue, M.J., 2013. Identifying hidden rate changes in the evolution of a binary morphological character: the evolution of plant habit in campanulid angiosperms. Syst. Biol. 62, 725–737. Bell, M.A., Lloyd, G.T., 2015. strap: an R package for plotting phylogenies against stratigraphy and assessing their stratigraphic congruence. Palaeontology 58, 379–389. Berland, L., 1922. Araignées. In: Voyage de M. le Baron de Rothschild en Ethiopie et en Afrique orientale anglaise (1904-1905): Résultats scientifiques: Animeux articulés. Paris 1, 43-90. Bertkau, P., 1872. Über die Respirationsorgane der Araneen. Arch. Nat., 38, 208-233. Blackledge, T.A., Scharff, N., Coddington, J.A., Szuts, T., Wenzel, J.W., Hayashi, C.Y., Agnarsson, I., 2009. Reconstructing web evolution and spider diversification in the molecular era. P. Natl. Acad. Sci. U.S.A. 106, 5229–5234. Blackwall, J., 1862. Descriptions of newly-discovered spiders from the island of Madeira. Ann. Mag. Nat. Hist. 9, 370–382. Bouckaert, R., Vaughan, T.G., Barido-Sottani, J., Duchêne, S., Fourment, M., Gavryushkina, A., Heled, J., Jones, G., Kühnert, D., De Maio, N., Matschiner, M., 2019. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Comput. Biol. 15, e1006650. Colgan, D.J., McLauchlan, A., Wilson, G.D.F., Livingston, S.P., Edgecombe, G.D., Macaranas, J., Cassis, G., Gray, M.R., 1998. Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Aust. J. Zool. 46, 419–437. Clerck, C., 1757.. Aranei Svecici. Svenska spindlar, uti sina hufvud-slågter indelte samt under några och sextio särskildte arter beskrefne och med illuminerade figurer uplyste. Stockholmiae 154 pp. Crews, S.C., Gillespie, R.G., 2010. Molecular systematics of Selenops spiders (Araneae: Selenopidae) from North and Central America: implications for Caribbean biogeography. Biol. J. Linn. Soc. 101, 288–322. Croeser, P.M.C., 1996. A revision of the African huntsman spider genus Palystes L. Koch 1875 (Araneae: Heteropodidae). Ann. Natal Mus. 37, 1–122. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. Davies, V.T., 1994. The huntsman spiders Heteropoda Latreille and Yiinthi gen. nov. (Araneae: Heteropodidae) in Australia. Mem. Queensland Mus. 35, 75–122. Doleschall, L., 1857. Bijdrage tot de kennis der Arachniden van den Indischen Archipel. Natuurkundig Tijdschrift voor Nederlandsch-Indie 13, 339–434. Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7 Mol. Biol. Evol. 29, 1969–1973. Dyal, S., 1957. A new genus of the spiders of the family Sparassidae. Res. B. Panjab Uni. Zool. 134, 561–566. Fernández, R., Kallal, R.J., Dimitrov, D., Ballesteros, J.A., Arnedo, M.A., Giribet, G., Hormiga, G., 2018. Phylogenomics, diversification dynamics, and comparative transcriptomics across the spider tree of life. Curr. Biol. 28, 1489–1497. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech. 3, 294–299. Franganillo, B.P., 1931. Excursiones arachnológicas, durante el mes de agosto de 1930. Estudios de “Belen” 25, 168-171. Garrison, N.L., Rodriguez, J., Agnarsson, I., Coddington, J.A., Griswold, C.E., Hamilton, C.A., Hedin, M., Kocot, K.M., Ledford, J.M., Bond, J.E., 2016. Spider phylogenomics: untangling the Spider Tree of Life. PeerJ 4, e1719. Geneious 2020.0.5. (https://www.geneious.com). Gertsch, W.J., 1933. Notes on American spiders of the family Thomisidae. Am. Mus. Nov. 593, 1–22. Gorneau, J.A., Rayor, L.S., Rheims, C.A., & Moreau, C.S., In Review. Molecular, morphological, and life history data to support research of huntsman spiders (Araneae: Sparassidae). Data in Brief. Hamilton-Attwell, V.L., Dippenaar-Schoeman, A.S., 2020. Observations on the egg-sacs of four Palystes spp. In the Fernkloof Nature Reserve (Araneae: Sparassidae). South African National Surv. Arachnida (SANSA) 35, 11–14. Hedin, M.C., Maddison, W.P., 2001. A Combined Molecular Approach to Phylogeny of the Jumping Spider Subfamily Dendryphantinae (Araneae: Salticidae). Mol. Phylogenet. Evol. 18, 386–403. Heibl C., 2008. PHYLOCH: R language tree plotting tools and interfaces to diverse phylogenetic software packages. http://www.christophheibl.de/Rpackages.html. Hentz, N.M., 1832. On North American spiders. Silliman’s J. Sci. Art. 21, 99–122. Hentz, N.M., 1844. Descriptions and figures of the araneides of the United States. Boston J. Nat. His. 4, 386–396. Hirst, A.S., 1911. The Araneae, Opiliones and Pseudoscorpiones. In: Percy Sladen Trust Expedition to the Indian Ocean in 1905 under the leadership of Mr. J. Stanley Gardiner. T. Linn. Soc. Lond. Zool., 2, 14, 379-395. Hirst, D.B., 1989. A new genus of huntsman spider (Heteropodidae: Araneae) from south eastern Australia. T. Roy. Soc. South. Aust. 113, 7–13. Hirst, D.B., 1990. A review of the genus Isopeda L. Koch (Heteropodidae: Araneae) in Australasia with descriptions of two new genera. Rec. So. Aust. Mus. 24, 11–26. Hirst, D.B., 1991a. Revision of the Australian genera Eodelena Hogg and Zachria L. Koch (Heteropodidae: Araneae). Rec. So. Aust. Mus. 25, 1–17. Hirst, D.B., 1991b. Revision of Australian species of the genus Holconia Thorell (Heteropodidae: Araneae. Rec. So. Aust. Mus. 24, 91–109. Hirst, D.B., 1992. Revision of the genus Isopeda Kock (Heteropodidae: Araneae) in Australia. Invertebr. Taxon. 6, 337–387. Hirst, D.B., 1993. Revision of the genus Isopedella (Araneae: Heteropodidae) Invertebr. Taxon 7, 33–87. Hirst, D.B., 1998. Irileka, a new heteropodine genus (Araneae: Heteropodidae) from Western Australia. Rec. West. Aust. Mus. 19, 141–144. Hoang, D.T., Chernomor, O., Von Haeseler, A., Minh, B.Q., Vinh, L.S., 2018. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522. Hogg, H.R., 1903. On the Australasian spiders of the subfamily Sparassinae. P. Zool. Soc. Lond., 72(II, 2, for 1902), 414-466. Hogg, H.R., 1914. Spiders collected by the Wollaston and British Ornithological Union Expeditions in Dutch New Guinea. P. Zool. Soc. Lond., 1914(C, Abstracts), 56-58. Holmberg, E.L., 1875. Descriptions et notices d’arachnides de la République Argentine. Period. Zool. 1, 283–302. Hsieh, Y.-L., Linsenmair, K.E., 2011. Underestimated spider diversity in a temperate beech forest. Biodivers. Conserv. 20, 2953–2965. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754–755. Huelsenbeck, J.P., Neilsen, R., Bollback, J.P., 2003. Stochastic mapping of morphological characters. Syst. Biol. 52, 131–138. Jäger, P., 1998. First results of a taxonomic revision of the SE Asian Sparassidae (Araneae). In: Selden, P.A. (Ed.), Proceedings of the 17th European Colloquium of Arachnology, Edinburgh, pp. 53–59. Jäger, P., 1999a. Sparassidae-the valid scientific name for the huntsman spiders (Arachnida: Araneae). Arachnologische Mitteilungen. 17, 1–10. Jäger, P., 1999b. Sinopoda, a new genus of Heteropodinae (Araneae, Sparassidae) from Asia. J. Arachnol. 27, 19–24. Jäger, P., 2000. Two new heteropodine genera from southern continental Asia (Araneae: Sparassidae). Acta Arachnol. 49, 61–71. Jäger, P., 2005a. A ‘swimming’ Heteropoda species from Borneo (Araneae, Sparassidae, Heteropodinae). J. Arachnol. 33, 715–718. Jäger, P., 2005b. New large-sized cave-dwelling Heteropoda species from Asia, with notes on their relationships (Araneae: Sparassidae: Heteropodinae). Rev. Suisse. Zool. 112, 87–114. Jäger, P., Vedel, V., 2005. Heteropoda dagmarae sp. nov. from Laos — a close relative of Heteropoda javana (Simon 1880) from Indonesia (Arachnida: Araneae: Sparassidae). Zootaxa 1044, 17–26. Jäger, P., 2006. Martensopoda gen. nov. from southern Indian mountain ranges, the first genus of huntsman spiders with a cymbial spur (Araneae: Sparassidae: Heteropodinae). Zootaxa 1325, 335–345. Jäger, P., Rheims, C.A., 2008. On the genera Origes Simon 1897, Prusias O. Pickard- Cambridge 1892, Tibellomma Simon 1903 and Paenula Simon 1897 from South and Central America. Senckenberg. Biol. 88, 29–39. Jäger, P., Praxaysombath, B., 2011. Selenops muehlmannorum spec. nov. from southern Laos (Araneae: Selenopidae). Zootaxa 2883, 65–68. Jäger, P., Krehenwinkel, H., 2015. May gen. n. (Araneae: Sparassidae): a unique lineage from southern Africa supported by morphological and molecular features. Afr. Invertebr. 56, 365–392. Jäger, P., 2020a. The spider genus Olios Walckenaer, 1837 (Araneae: Sparassidae)—Part 1: species groups, diagnoses, identification keys, distribution maps and revision of the argelasius-, coenobitus-and auricomis-groups. Zootaxa 4866, 1–119. Jäger, P., 2020b. Thunberga gen. nov., a new genus of huntsman spiders from Madagascar (Araneae: Sparassidae: Heteropodinae). Zootaxa 4790, 245–260. Jäger, P., 2020c. Platnickopoda gen. nov., a new genus of huntsman spiders from Tanzania (Araneae: Sparassidae: Heteropodinae). Arachnology 18, 499–506. Jäger, P., 2021. Two new enigmatic genera of huntsman spiders from Madagascar (Araneae: Sparassidae). Zootaxa 4984, 335–346. Järvi, T.H., 1912. Das Vaginalsystem der Sparassiden. I. Allgemeiner Teil. Ann. Acad. Sci. Fenn. (A) 4: 1-131, pl. 1-11. Jones, C., Rayor, L., 2017. Retreat availability and social influences on retreat sharing in group-living huntsman spiders, Delena lapidicola and Delena cancerides (Araneae: Sparassidae). J. Arachnol. 45, 271–276. Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K., von Haeseler, A., Jermiin, L.S., 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods. 14, 587. Kammerer, C.F., 2006. Notes on some preoccupied names in Arthropoda. Acta Zootaxonomica Sinica 31, 269–271. Karsch, F., 1880. Arachnologische Blätter (Decas I). Z. Ges. Nat. Dritte Folge 5, 373–409. Karsch, F., 1884. Arachnoidea. In: Greeff, R. (ed.) Die Fauna der Guinea-Inseln S.-Thomé und Rolas. Sitzber. G. zur Beförderung der Gesamten Nat. zu Marburg, 2, 60-68, 79. Katoh, K., Standley, D.M., 2013. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30, 772–780. J.A. Gorneau et al. http://refhub.elsevier.com/S1055-7903(22)00143-9/h0020 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0020 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0020 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0025 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0025 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0025 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0030 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0030 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0035 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0035 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0035 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0040 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0040 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0045 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0050 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0050 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0050 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0055 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0055 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0060 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0065 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0065 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0065 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0070 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0070 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0070 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0080 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0080 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0080 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0085 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0085 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0090 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0090 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0090 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0090 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0095 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0095 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0095 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0100 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0100 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0100 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0105 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0105 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0105 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0110 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0110 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0115 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0115 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0120 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0120 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0125 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0125 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0130 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0130 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0135 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0135 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0140 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0140 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0140 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0145 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0145 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0145 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0155 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0155 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0155 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0165 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0165 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0175 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0175 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0175 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0180 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0180 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0180 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0190 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0195 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0195 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0205 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0205 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0210 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0210 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0215 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0215 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0220 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0220 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0225 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0225 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0230 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0230 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0235 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0235 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0240 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0240 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0255 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0255 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0260 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0260 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0265 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0265 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0270 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0270 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0275 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0275 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0275 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0280 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0280 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0285 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0285 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0290 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0290 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0295 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0295 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0300 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0300 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0300 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0305 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0305 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0305 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0310 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0310 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0310 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0315 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0315 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0315 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0320 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0320 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0325 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0325 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0325 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0330 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0330 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0330 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0335 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0335 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0340 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0340 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0345 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0345 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0355 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0355 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0355 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0360 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0360 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0360 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0365 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0365 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0370 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0380 http://refhub.elsevier.com/S1055-7903(22)00143-9/h0380 Molecular Phylogenetics and Evolution 174 (2022) 107530 14 Keyserling, E., 1884. Neue Spinnen aus America. V. Verhandlungen der Kaiserlich- Königlichen Zoologisch-Botanischen Gesellschaft in Wien 33, 649-684. Koch, C.L. & Berendt, G.C. 1854. Die im Bernstein befindlichen Myriapoden, Arachniden und Apteren der Vorwelt. In Berendt, G. C. Die in Bernstein befindlichen organischen Reste der Vorwelt gesammelt in verbindung mit mehreren bearbeitetet und herausgegeben 1. Berlin, Nicolai, 124 pp. Koch, L., 1867. Beschreibungen neuer Arachniden und Myriapoden. II. Verhandlungen der Kaiserlich-Königlichen Zoologisch-Botanischen Gesellschaft in Wien 17, 173–250. Koch, L., 1875. Die Arachniden Australiens, nach der Natur beschrieben und abgebildet [Erster Theil, Lieferung 12–16]. Bauer & Raspe, Nürnberg 577–740, 44–65. Koch, L., 1876. Die Arachniden Australiens, nach der Natur beschrieben und abgebildet [Erster Theil, Lieferung 17–19]. Bauer & Raspe, Nürnberg 741–888, 65–76. Kolosváry, G., 1934. Neue Spinnen aus Australien und Neu-Guinea. Folia Zool. Hydrobiol., Rigā, 7, 44-48, f. 1-11. Latreille, P.A., 1804. Tableau methodique des Insectes. Nouveau Dictionnaire d’Histoire Naturelle, Paris 24, 129–295. Latreille, P. A., 1806. Genera crustaceorum et insectorum. Paris, 1, 82-127. Latreille, P.A., 1809. Genera crustaceorum et insectorum. Paris, 4, 370-371. Lawrence, R.F., 1928. Contributions to a knowledge of the fauna of South-West Africa VII. Arachnida, Part 2. Ann. South Afr. Mus. 25, 217–312. Lawrence, R.F., 1937. A collection of Arachnida from Zululand. Ann Natal Mus. 8, 211–273. Lawrence, R.F., 1938. Transvaal Museum Expedition to South-West Africa and Little Namaqualand, May to August 1937. Spiders. Ann. Transvaal Mus. 19, 215–226. Lawrence, R.F., 1962. Spiders of the Namib desert. Ann. Transvaal Mus. 24, 197–211. Lawrence, R.F., 1965. New and little known Arachnida from the Namib desert, S.W. Africa. Sci. P. Namib. Des. Res. Station, 27, 1-12. Leardi in Airaghi, Z., 1901. Aracnidi di Almora. Atti della Società Italiana di Scienze Naturali e del Museo Civico di Storia Naturale di Milano 40, 85-94. de Lessert, R., 1921. Araignées du Kilimandjaro et du Merou (suite). 4. Clubionidae. Rev. Suisse Zool. 28, 381–442. Maddison, W.P., Maddison, D.R., 2018. Mesquite: a modular system for evolutionary analysis. http://mesquiteproject.org. Version 3.6. Magalhães, I.L., Azevedo, G.H., Michalik, P., Ramírez, M.J., 2020. The fossil record of spiders revisited: implications for calibrating trees and evidence for a major faunal turnover since the Mesozoic. Biol. Rev. 95, 184–217. Marx, G., 1889. On the new spider of the genus Dinopis, from the southern United States. P. Acad. Nat. Sci. Phila 41, 341–343. de Mello-Leitão, C.F., 1940. Aranhas do Espírito Santo coligidas por Mario Rosa, em 1936 e 1937. Arq. Zool. Estado de Sao Paulo 2, 199–214. Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. GCE Workshop. 1–8. Moradmand, M., Jäger, P., 2012. Taxonomic revision of the huntsman spider genus Eusparassus Simon, 1903 (Araneae: Sparassidae) in Eurasia. J. Nat. Hist. 46 (39–40), 2439–2496. Moradmand, M., Schönhofer, A.L., Jäger, P., 2014. Molecular phylogeny of the spider family Sparassidae with focus on the genus Eusparassus and notes on the RTA-clade and ‘Laterigradae’. Mol. Phylogenet. Evol. 74, 48–65. Moradmand, M., Zamani, A., Jäger, P., 2016. On the genus Cebrennus Simon, 1880 in Iran with description of a new species from Iranian Central Desert (Araneae: Sparassidae). Zootaxa. 4121, 187–193. Neuwirth, E., 2014. RColorBrewer: ColorBrewer Palettes. https://cran.r-project.org. Nguyen, L.-T., Schmidt, H.A., von Haeseler, A., Minh, B.Q., 2015. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Mol. Biol. Evol. 32, 268–274. Palumbi, S.R., Martin, A., Romano, S., McMillan, W.O, Stice, L., 1991. The Simple Fool’s Guide to PCR. Special Publication, Department of Zoology and Kewalo Marine Laboratory, Honolulu: University of Hawaii. Petrunkevitch, A., 1925. Arachnida from Panama. T. Conn. Acad. Art. Sci. 27, 51–248. Petrunkevitch, A., 1928. Systema Aranearum. T. Conn. Acad. Art. Sci. 29, 1–270. Petrunkevitch, A.I., 1950. Baltic amber spiders in the Museum of Comparative Zoology. B. Mus. Comparat. Zool. 103, 257–337. Petrunkevitch, A.I., 1958. Amber spiders in European collections. T. Conn. Acad. Art. Sci. 41, 97–400. Pickard-Cambridge, O., 1892. Arachnida. Araneida. In: Biologia Centrali-Americana, Zoology. London 1, 89–104. Pinto, E.C., Rheims, C.A., 2016. A new genus of Neotropical spiders of the family Sparassidae (Arachnida. Araneae). Zoologia (Curitiba) 33 (6, e20160160), 1–15. Plummer, M., Best, N., Cowles, K., Vines, K., 2006. CODA: Convergence Diagnosis and Output Analysis for MCMC. R News 6, 7–11. Pocock, R.I., 1898. The Arachnida from the regions of Lakes Nyassa and Tanganyika contained in the collection of the British Museum. Ann. Mag. Nat. Hist. 2, 429–448. Rainbow, W.J., 1899. Contribution to a knowledge of the araneidan fauna of Santa Cruz. P. Linn. Soc. N. S. W. 24, 304–321. Rainbow, W.J., 1917. Araneidae. In: Results of the South Australian Museum Expedition to Strzelecki and Coopers Creeks. T. P. Roy. Soc. South Aust., 41, 482-489. Rambaut, A., Drummond, A.J., Xie, D., Baele, G., Suchard, M.A., 2018. Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Syst. Biol. 67, 901–904. Rambaut, A., 2018. FigTree v. 1.4.4. http://tree.bio.ed.ac.uk/software/figtree/. Ramírez, M.J., 2014. The morphology and phylogeny of dionychan spiders (Araneae: Araneomorphae). B. Am. Mus. Nat. Hist. 390, 1–374. Revell, L.J., 2012. phytools: An R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, 217–223. https://doi.org/10.1111/j.2041- 210X.2011.00169.x. Rheims, C.A., 2007a. Análise cladística dos gêneros de Sparassidae Bertkau (Arachnida, Araneae). Tese (Doutorado) – Instituto de Biociências da Universidade de São Paulo, Departamento de Zoologia. Rheims, C.A., 2007b. Revision of the Neotropical spider genus Macrinus (Araneae, Sparassidae). J. Arachnol. 35, 159–170. Rheims, C.A., & Jäger, P., 2008. Revision of the Neotropical huntsman spider genus Vindullus Simon (Araneae, Sparassidae). J. Arachnol., 36, 221-231. Rheims, C.A., 2010a. Caayguara, a new genus of huntsman spiders from the Brazilian Atlantic forest (Araneae: Sparassidae). Zootaxa, 2630, 1-29. Rheims, C.A., 2010b. On the native Nearctic species of the huntsman spider family Sparassidae Bertkau (Araneae). J. Arachnol. 38, 530–537. Rheims, C.A., 2010c. Notes on the Neotropical genus Macrinus (Araneae: Sparassidae). Zoologia (Curitiba) 27, 440–444. Rheims, C.A., 2010d. A new genus of huntsman spiders from the Neotropical region (Araneae: Sparassidae: Heteropodinae). Zootaxa 2650, 33–46. Rheims, C., 2013. A new genus of huntsman spiders (Araneae, Sparassidae, Sparianthinae) from the Neotropical region. Zootaxa 3734, 199–220. Rheims, C.A., 2015. Curicaberis, a new genus of Sparassidae from North and Central America (Araneae, Sparassidae, Sparassinae). Zootaxa 4012, 401–446. Rheims, C.A., Alayón, G., 2016. Neostasina gen. nov., a new genus of huntsman spiders from the Neotropical region (Araneae, Sparassidae, Sparianthinae). Zootaxa 4079, 301–344. Rheims, C.A., Alayón, G., 2018. A new genus of minute Sparianthinae spiders from the Caribbean (Araneae, Sparassidae). ZooKeys 742, 13–22. Rheims, C.A., 2019a. On the huntsman spider genus Vindullus Simon, 1880 (Araneae: Sparassidae). Zootaxa 4544, 572–580. Rheims, C.A., 2019b. Extraordinarius gen. nov., a new genus of Sparianthinae spiders (Araneae: Sparassidae) from southeastern Brazil. Zootaxa 4674, 83–99. Rheims, C.A., Jäger, P., 2022. Revalidation of the genus Sadala Simon, 188