bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.185769; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Genomic resources and toolkits for developmental study of whip 2 spiders (Amblypygi) provide insights into arachnid genome evolution 3 and antenniform leg patterning 4 5 Guilherme Gainett1, Prashant P. Sharma1 6 7 1Department of Integrative Biology, University of Wisconsin-Madison, Madison, WI, USA 8 53706 9 Correspondence: [email protected] 10 11 Abstract 12 13 Background 14 The resurgence of interest in the comparative developmental study of chelicerates has led to 15 important insights, such as the discovery of a genome duplication shared by spiders and 16 scorpions, inferred to have occurred at the most recent common ancestor of 17 Arachnopulmonata (a clade comprised of the five book lung-bearing arachnid orders). 18 Nonetheless, several arachnid groups remain understudied in the context of development and 19 genomics, such as the order Amblypygi (whip spiders). The phylogenetic position of 20 Amblypygi in Arachnopulmonata posits them as an interesting group to test the incidence of 21 the proposed genome duplication in the common ancestor of Arachnopulmonata, as well as 22 the degree of retention of duplicates over 450 Myr. Moreover, whip spiders have their first 23 pair of walking legs elongated and modified into sensory appendages (a convergence with the 24 antenna of mandibulates), but the genetic patterning of these antenniform legs has never been 25 investigated. 26 27 Results 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.185769; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 28 We established genomic resources and protocols for cultivation of embryos and gene 29 expression assays by in situ hybridization to study the development of the whip spider 30 Phrynus marginemaculatus. Using embryonic transcriptomes from three species of 31 Amblypygi, we show that the ancestral whip spider exhibited duplications of all ten Hox 32 genes. We deploy these resources to show that paralogs of the leg gap genes dachshund and 33 homothorax retain arachnopulmonate-specific expression patterns in P. marginemaculatus. 34 We characterize the expression of leg gap genes Distal-less, dachshund-1/2 and homothorax- 35 1/2 in the embryonic antenniform leg and other appendages, and provide evidence that 36 allometry, and by extension the antenniform leg fate, is specified early in embryogenesis. 37 38 Conclusion 39 This study is the first step in establishing P. marginemaculatus as a model for modern 40 chelicerate evolutionary developmental study, and provides the first resources sampling whip 41 spiders for comparative genomics. Our results suggest that Amblypygi share a genome 42 duplication with spiders and scorpions, and set up a framework to study the genetic 43 specification of antenniform legs. Future efforts to study whip spider development must 44 emphasize the development of tools for functional experiments in P. marginemaculatus. 45 46 Keywords: Arachnida | Arachnopulmonata | gene duplication | paralogs | leg gap genes | 47 sensory biology 48 49 Background 50 51 In the past 25 years, comparative developmental study of chelicerates has resurged with the 52 integration of molecular biology and genomics, spearheaded by developmental genetic 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.185769; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 53 investigations of the spiders Cupiennius salei and Parasteatoda tepidariorum [1-6]. Such 54 works have revealed several important aspects of chelicerate development, such as the 55 molecular mechanism of dorso-ventral axis patterning [3,7-9], segmentation of the prosoma 56 [1,10-13] and opisthosoma [2,13], and specification of prosomal versus opisthosomal fate 57 [14,15]. A few non-spider chelicerate models have also emerged in recent years and provided 58 new perspectives on chelicerate development, such as the horseshoe crab Limulus 59 polyphemus (Xiphosura) [16,17] , the mites Archegozetes longisetosus [18-21] and 60 Tetranychus urticae (Acariformes) [22-24], the tick Rhipicephalus microplus (Parasitiformes) 61 [25], the Arizona bark scorpion Centruroides sculpturatus (Scorpiones) [26] and the 62 harvestmen Phalangium opilio (Opiliones) [27-30]. Studies in A. longisetosus and C. 63 sculpturatus have suggested that changes in Hox gene number and expression domains are 64 responsible for such phenomena as the reduced segmentation of mites and the supernumerary 65 posterior appendage identities of scorpions [18,26]. 66 67 In addition to the insights into chelicerate development, one of the major recent 68 outcomes of increasing availability of genomic resources in in the group was the discovery of 69 a whole or partial genome duplication in spiders and scorpions. This genome duplication is 70 inferred to trace back to the most recent common ancestor of the recently proposed clade 71 Arachnopulmonata, which includes spiders, scorpions, and three other arachnid orders with 72 book lungs [6,26,31-33]. This duplication event is also inferred to be independent from the 73 multiple rounds of whole genome duplication undergone by horseshoe crabs (Xiphosura) 74 [34,35]. The duplication in spiders and scorpions encompasses several important 75 developmental genes, such as the homeobox family [6,32]. Excitingly, there is increasing 76 evidence of divergent expression and function of paralogs, such as in the case of Hox genes, 77 leg gap genes and Retinal Determination Gene Network homologs [6,26,36-40]. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.185769; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 78 79 Nevertheless, knowledge on the development of several arachnid orders still remains scarce 80 or entirely unexplored in the contexts of embryological study and genomic architecture. One 81 particularly interesting arachnid order whose genomic evolution and developmental biology 82 remains largely unexplored is Amblypygi. Commonly known as whip spiders, Amblypygi 83 comprise approximately 220 described species of nocturnal predators that are distributed 84 primarily in the tropics and subtropics worldwide [41,42]. Amblypygi is part of the clade 85 Pedipalpi (together with the orders Thelyphonida and Schizomida), which in turn is sister 86 group to spiders (Araneae) [31,43-45]. Considering the phylogenetic position of Amblypygi 87 within Arachnopulmonata, a better understanding of Amblypygi genomics is anticipated to 88 facilitate exploration of the evolutionary outcomes of the genome duplication in 89 Arachnopulmonata, and better characterizing the extent to which arachnopulmonate orders 90 retained and specialized the ensuing paralogous genes. Unfortunately, the embryology of the 91 order Amblypygi is known only from the works of Pereyaslawzewa [46], a brief mention in 92 Strubell [47] (“Phrynidae”), Gough [48], and the comprehensive study of Weygoldt [49] in 93 Phrynus marginemaculatus (formerly Tarantula marginemaculata). 94 95 Beyond the interest in their genomic architecture, whip spiders possess a fascinating biology 96 and natural history. Whip spiders are emerging as model organisms in behavioral ecology, 97 learning, and neurophysiology in arachnids [50,51]. Their pedipalps, the second pair of 98 appendages, are robust raptorial devices used for striking prey, including mostly arthropods, 99 and even small vertebrates [41,50,52]. However, their most conspicuous characteristic and 100 namesake is their “whip”, a modified antenniform first walking leg that is not used for 101 locomotion, but instead as a sensory appendage. The antenniform legs are extremely 102 elongated relative to the other leg pairs, and the tibia and tarsus are pseudo-segmented into 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.03.185769; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 103 hundreds of articles that harbor an array of chemo-, thermo-, hygro- and mechanoreceptive 104 sensilla [53-55]. The peripheral circuitry of the antenniform legs is complex, exhibiting 105 peripheral synapses and giant neurons which are possibly involved in fast sensory responses 106 [55-57]. The antenniform legs also have been shown to be important for foraging, mating, 107 and intrasexual contests [58,59]. Notably, concentration of sensory structures, elongation and 108 pseudo-segmentation of these legs constitute a striking convergence with the antenna of 109 mandibulate arthropods (Pancrustacea and Myriapoda). Evidence from Hox gene expression 110 and functional experiments support the view that the antenna of mandibulates is positionally 111 homologous to the chelicera of chelicerates (deutocerebral appendage) [21,30]. Despite their 112 different positions along the antero-posterior axis of the body, the serial homology of 113