bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

Widespread retention of ohnologs in key developmental gene families

following whole genome duplication in arachnopulmonates

Amber Harper1, Luis Baudouin Gonzalez1, Anna Schönauer1, Ralf Janssen2, Michael

Seiter3, Michaela Holzem1,4, Saad Arif1,5, Alistair P. McGregor1,5*, Lauren Sumner-

Rooney6,*

1Department of Biological and Medical Sciences, Faculty of Health and Life

Sciences, Oxford Brookes University, Oxford, OX3 0BP, United Kingdom.

2Department of Earth Sciences, Uppsala University, Geocentrum, Villavägen 16, 752

36 Uppsala, Sweden.

3Department of Evolutionary Biology, Unit Integrative Zoology, University of Vienna,

Althanstrasse 14, 1090 Vienna, Austria.

4Division of Signalling and Functional Genomics, German Cancer Research Centre

(DKFZ), Heidelberg, Germany and Department of Cell and Molecular Biology,

Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.

5Centre for Functional Genomics, Oxford Brookes University, Oxford, OX3 0BP,

United Kingdom.

6Oxford University Museum of Natural History, University of Oxford, Oxford OX1

3PW, United Kingdom.

*Corresponding authors: [email protected] (APM) and lauren.sumner-

[email protected] (LSR).

1 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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.

ABSTRACT

Whole genome duplications have occurred multiple times during animal evolution,

including in lineages leading to vertebrates, teleosts, horseshoe crabs and

arachnopulmonates. These dramatic events initially produce a wealth of new genetic

material, generally followed by extensive gene loss. It appears, however, that

developmental genes such as homeobox genes, signalling pathway components and

microRNAs are frequently retained as duplicates (so called ohnologs) following

whole-genome duplication. These not only provide the best evidence for whole-

genome duplication, but an opportunity to study its evolutionary consequences.

Although these genes are well studied in the context of vertebrate whole-genome

duplication , similar comparisons across the extant arachnopulmonate orders are

patchy. We sequenced embryonic transcriptomes from two spider species and two

amblypygid species and surveyed three important gene families, Hox, Wnt and

frizzled, across these and twelve existing transcriptomic and genomic resources for

chelicerates. We report extensive retention of putative ohnologs, including

amblypygids, further supporting the ancestral arachnopulmonate whole-genome

duplication. We also find evidence of consistent evolutionary trajectories in Hox and

Wnt gene repertoires across three of the five arachnopulmonate orders, with inter-

order variation in the retention of specific paralogs. We identify variation between

major clades in spiders and are better able to reconstruct the chronology of gene

duplications and losses in spiders, amblypygids, and scorpions. These insights shed

light on the evolution of the developmental toolkit in arachnopulmonates, highlight

the importance of the comparative approach within lineages, and provide substantial

new transcriptomic data for future study.

Keywords

2 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Arachnids, developmental toolkit, gene duplication, transcriptomes, whole genome

duplication.

INTRODUCTION

The duplication of genetic material is an important contributor to the evolution of

morphological and physiological innovations (Ohno 1970; Zhang 2003). The most

dramatic example of this is whole genome duplication (WGD), when gene copy

numbers are doubled and retained paralogs (ohnologs) can then share ancestral

functions (subfunctionalization) and/or evolve new roles (neofunctionalization) (Ohno

1970; Force et al. 1999; Lynch and Conery 2000). The occurrence of two rounds

(2R) of WGD in the early evolution of vertebrates has long been associated with their

taxonomic and morphological diversity (e.g. Ohno 1970; Holland et al. 1994; Dehal

and Boore 2005; Holland 2013), and a subsequent 3R in teleosts is frequently linked

to their success as the most diverse vertebrate group (e.g. Meyer and Schartl 1999;

Glasauer and Neuhauss 2014). However, this remains controversial and difficult to

test (Donoghue and Purnell 2005) and in several animal lineages there is no clear

association between WGD and diversification (Mark Welch et al. 2008; Flot et al.

2013; Havlak et al. 2014; Kenny et al. 2016; Nong et al. 2020). Along with

vertebrates, chelicerates also appear to be hotspots of WGD, with up to three rounds

reported in horseshoe crabs (Havlak et al. 2014; Kenny et al. 2016; Nong et al.

2020), one in the ancestor of arachnopulmonates (spiders, scorpions, and their

allies) (Schwager et al. 2017), and potentially two further rounds within the spider

clade Synspermiata (Král et al. 2019). Chelicerates demonstrate a highly variable

body plan, occupy a wide range of habitats and ecological niches, and have evolved

a variety of biologically important innovations such as venoms and silks (Schwager

et al. 2015). They therefore offer an excellent opportunity for comparison with

3 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

vertebrates concerning the implications of WGD for morphological and taxonomic

diversity, and genome evolution in its wake.

The house spider Parasteatoda tepidariorum has emerged as a model species to

study the impacts of WGD on arachnid evolution and development. Genomic and

functional developmental studies have found retained ohnologs of many important

genes, with evidence for neo- and subfunctionalization compared to single-copy

orthologs in arachnids lacking WGD (Janssen et al. 2015; Leite et al. 2016; Turetzek

et al. 2016; Schwager et al. 2017; Turetzek et al. 2017; Leite et al. 2018; Baudouin-

Gonzalez et al. 2021). Work on the scorpions Centruroides sculpturatus and

Mesobuthus martensii has consistently complemented findings in P. tepidariorum,

with genomic studies recovering many ohnologs retained in common with spiders (Di

et al. 2015; Sharma et al. 2015; Leite et al. 2018). In the past few years, several

additional spider genomes have become available, providing an opportunity to get a

more detailed view of genome evolution following WGD. Although synteny analysis

remains the gold standard for the identification of ohnologs, the required

chromosome-level genomic assemblies remain relatively scarce. Work on the P.

tepidariorum, C. sculpturatus and M. martensii genomes has been complemented by

targeted studies of individual gene families and transcriptomic surveys (Schwager et

al. 2007; Sharma et al. 2012; Leite et al. 2018; Gainett and Sharma 2020).

Combined with phylogenetic analyses, the identification of duplications can provide

evidence of WGD events and their timing in arachnid evolution. Although

transcriptomes can yield variant sequences of individual genes, from different alleles

or individuals in mixed samples, these are generally straight-forward to filter out from

truly duplicated loci owing to substantial sequence divergence in the latter. They also

offer the double-edged sword of capturing gene expression, rather than presence in

4 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

the genome; pseudogenized or silenced duplicates are not detected, but neither are

functional genes if they are not expressed at the sampled timepoint or tissue. Such

studies have produced strong additional evidence for an ancestral WGD, with

patterns of duplication coinciding with our expectations for arachnopulmonate

ohnologs (Clarke et al. 2014; Clarke et al. 2015; Sharma et al. 2015; Turetzek et al.

2017; Leite et al. 2018; Gainett and Sharma 2020; Gainett et al. 2020).

Comparison of WGD events among arachnopulmonates, horseshoe crabs and

vertebrates indicates that despite extensive gene loss following duplication events,

certain gene families are commonly retained following duplication (Holland et al.

1994; Schwager et al. 2007; Kuraku and Meyer 2009; Di et al. 2015; Sharma et al.

2015; Kenny et al. 2016; Leite et al. 2016; Schwager et al. 2017; Leite et al. 2018).

These typically include genes from the conserved developmental ‘toolkit’ of

transcription factors (TFs), cell signalling ligands and receptors, and microRNAs

(Erwin 2009). Among these, several have stood out as focal points in the study of

gene and genome duplications. The Hox group of homeobox genes regulate the

identity of the body plan along the antero-posterior axis of all bilaterian animals

(McGinnis and Krumlauf 1992; Abzhanov et al. 1999; Carroll et al. 2005; Pearson et

al. 2005; Hueber and Lohmann 2008; Holland 2013). Four clusters of these key

developmental genes were retained after 1R and 2R in vertebrates (Holland et al.

1994; Meyer and Schartl 1999; Kuraku and Meyer 2009; Pascual-Anaya et al. 2013),

and the arachnopulmonate WGD is evident in the almost universal retention of Hox

gene duplicates in sequenced genomes, with two ohnologs of all ten arthropod Hox

genes in the scorpion M. martensii (Di et al. 2015; Leite et al. 2018), all except Hox3

being represented by two copies in C. sculpturatus (Leite et al. 2018), and all except

fushi tarazu (ftz) in P. tepidariorum (Schwager et al. 2017). Systematic studies of

5 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Hox gene expression patterns in the latter demonstrated that all nine pairs of Hox

paralogs exhibit signs of sub- or neofunctionalization (Schwager et al. 2017). This

high level of retention and expression divergence lends strong support to the

importance of Hox gene duplication in the evolution of the arachnopulmonate body

plan, and further consolidates the position of this family as a key indicator of WGD.

Additionally, arachnids lacking WGD, such as ticks, mites, and harvestmen, exhibit

single copies of the Hox genes, with no evidence for duplication via other routes

(Grbić et al. 2011; Leite et al. 2018; Gainett et al. 2021).

In addition to TFs, the ligands and receptors of some signalling pathways of the

developmental toolkit (e.g. Hedgehog, Wnt, TGF-ß, NHR) also demonstrate higher

copy numbers in vertebrates and other groups subject to WGD, including

arachnopulmonates (Holland et al. 1994; Meyer and Schartl 1999; Shimeld 1999;

Pires-daSilva and Sommer 2003; Cho et al. 2010; Janssen et al. 2010; Hogvall et al.

2014; Janssen et al. 2015). The Wnt signalling pathway plays many important roles

during animal development, including segmentation and patterning of the nervous

system, eyes and gut (Erwin 2009; Murat et al. 2010). In the canonical pathway, Wnt

ligands bind to transmembrane receptors, such as Frizzled, to trigger translocation of

ß-catenin to the nucleus and mediate regulation of gene expression (Cadigan and

Nusse 1997; Hamilton et al. 2001; Logan and Nusse 2004; van Amerongen and

Nusse 2009). There are thirteen subfamilies of Wnt genes found in bilaterians, as

well as multiple receptor families and downstream components. In contrast to the

extensive retention of Hox ohnologs following WGD, Wnt duplicates in P.

tepidariorum appear to be restricted to Wnt7 and Wnt11, with the remaining eight

subfamilies represented by single genes (Janssen et al. 2010). However, these are

the only reported Wnt gene duplications in arthropods despite several recent surveys

6 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

(Bolognesi et al. 2008; Murat et al. 2010; Hayden and Arthur 2013; Meng et al. 2013;

Hogvall et al. 2014; Janssen and Posnien 2014; Holzem et al. 2019), and beyond P.

tepidariorum no other arachnopulmonates have been systematically searched.

Similarly, duplications within the four frizzled gene subfamilies appear to be

restricted to arachnopulmonates among arthropods, wherein only fz4 is duplicated in

both P. tepidariorum and M. martensii (Janssen et al. 2015).

Several Wnt families have also been retained after the 1R and 2R events in

vertebrates, for example there are two copies each of Wnt2, Wnt3, Wnt5, Wnt7,

Wnt8, Wnt9, and Wnt10 in humans (Miller 2001; Janssen et al. 2010). However, no

subfamilies are represented by three or four copies in humans and so there is some

consistency with arachnopulmonates in that the Wnts may be more conservative

markers of WGD, to be used in combination with Hox and other homeobox genes.

The extensive and consistent retention of key developmental genes like Hox genes

apparent in P. tepidariorum and C. sculpturatus, and Wnt genes in P. tepidariorum,

strongly support the occurrence of an ancestral WGD in arachnopulmonates.

However, data are only available for a handful of species so far, resulting in very

patchy taxonomic sampling. For example, only P. tepidariorum and Pholcus

phalangioides have been comprehensively surveyed for homeobox genes among

spiders (Leite et al. 2018), omitting the large and derived retrolateral tibial apophysis

(RTA) clade, which includes jumping spiders, crab spiders and other free hunters,

and the systematic identification of Wnt genes has been restricted to only P.

tepidariorum. Spiders and scorpions are by far the most speciose of the

arachnopulmonates, and there may be additional diversity in their repertoires of

these important developmental gene families of which we are not yet aware.

7 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

In addition, and perhaps more urgently, only two of the five arachnopulmonate

lineages have dominated the field thus far; sufficient genomic information for

comparison is lacking beyond spiders and scorpions. Also represented in

Arachnopulmonata are the amblypygids (whip spiders), relatively understudied and

enigmatic animals comprising around 190 extant species. They exhibit highly derived

morphology of the pedipalps, which are adapted to form raptorial appendages, and

of the first pair of walking legs, which are antenniform and can comprise more than

100 segments (Weygoldt 2009). Despite the scarcity of transcriptomic or genomic

data for amblypygids (whip spiders) (Gainett and Sharma 2020; Gainett et al. 2020

for recent advances), their widely accepted position within Arachnopulmonata

implies that they were also subject to an ancestral WGD. A recent survey of the

Phrynus marginemaculatus transcriptome supported this in the recovery of multiple

duplicate Hox and leg gap genes (Gainett and Sharma 2020). Particularly given the

derived nature of their appendages, this group could shed substantial light on

genomic and morphological evolution following WGD.

To better understand the genomic consequences of WGD in a greater diversity of

arachnopulmonate lineages, we sequenced de novo embryonic transcriptomes from

two spiders belonging to the derived RTA clade and two amblypygids. We surveyed

Hox, Wnt and frizzled genes in these species and existing genomic and

transcriptomic resources for comparison with other arachnids, both with and without

an ancestral WGD, improving sampling at both the order and sub-order levels.

MATERIALS AND METHODS

Embryo collection, fixation and staging

8 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Embryos of mixed ages were collected from captive females of the amblypygids

Charinus acosta (Charinidae; parthenogenetic, collected at one day, one month and

two months after the appearance of egg sacs; equivalent to approximately 1%, 30%

and 60% of development) and Euphrynichus bacillifer (Neoamblypygi: Phrynichidae;

mated, collected at approximately 30% of development), the wolf spider Pardosa

amentata (collected in Oxford, UK, equivalent to stages 11/12 in P. tepidariorum)

and mixed stage embryos of the jumping spider Marpissa muscosa (kindly provided

by Philip Steinhoff and Gabriele Uhl) and stored in RNAlater. Phalangium opilio were

collected in Uppsala, Sweden, and developmental series of embryos ranging from

egg deposition to the end of embryogenesis were collected for sequencing.

Transcriptomics

Total RNA was extracted from mixed aged embryos, pooled by species, of Charinus

acosta, Euphrynichus bacillifer, Pardosa amentata and Marpissa muscosa using

QIAzol lysis reagent according to the manufacturer’s instructions (Qiagen). Illumina

libraries were prepared using a TruSeq RNA kit (including polyA selection) and

sequenced on the NovaSeq platform (100bp PE, Edinburgh Genomics). The quality

of raw reads was assessed using FastQC v0.11.9 (Andrews 2010), erroneous k-

mers were corrected using rCorrector (default settings; Song and Florea 2015), and

unfixable read pairs (from low-expression homolog pairs or containing too many

errors) were discarded using a custom Python script (available at

https://github.com/harvardinformatics/TranscriptomeAssemblyTools/blob/master/Filte

rUncorrectabledPEfastq.py courtesy of Adam Freeman). Adapter sequences were

identified and removed and low quality ends (phred score cut-off = 5) trimmed using

TrimGalore! v0.6.5 (available at https://github.com/FelixKrueger/TrimGalore). De

novo transcriptome assembly was performed using only properly paired reads with

9 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Trinity v2.10.0 (Haas et al. 2013) using default settings. Transcriptome completeness

was evaluated on the longest isoform per gene using BUSCO v4.0.2 (77) along with

the arachnid database (arachnida_odb10 created on 2019-11-20; 10 species, 2934

BUSCOs) and the arthropod database (arthropoda_odb10 created on 2019-11-20;

90 species, 1013 BUSCOs). Reads are available on SRA (BioProject

PRJNA707377) and assembled transcriptomes are available at (XXXXX).

Identification of gene candidates

To identify Hox, Wnt and frizzled gene candidates across chelicerates, we performed

BLAST searches (p-value 0.05) against existing genomic and transcriptomic

resources and the four new embryonic transcriptomes generated in this study. Hox,

Wnt and Frizzled peptide sequences previously identified in P. tepidariorum and

Ixodes scapularis were reciprocally blasted against the respective NCBI proteomes

and the top hit was selected (Supplementary File 1) (Janssen et al. 2010; Janssen et

al. 2015; Schwager et al. 2017; Leite et al. 2018). Hox protein sequences previously

identified in C. sculpturatus (Schwager et al. 2017) and P. opilio (Leite et al. 2018)

were reciprocally blasted against the respective NCBI proteomes and the top hit was

selected (Supplementary File 1). These sequences along with the Hox and Fz

peptide sequences identified in M. martensii genome were used as query sequences

in our analysis (Di et al. 2015; Janssen et al. 2015).

BLASTP searches were performed against the NCBI proteomes of Stegodyphus

dumicola, C. sculpturatus, Tetranychus urticae and Limulus polyphemus. TBLASTN

searches were performed against C. acosta, E. bacillifer, P. amentata and M.

muscosa (this study); the transcriptomes of P. phalangioides (Turetzek, Torres-Oliva,

Kaufholz, Prpic, Posnien, in preparation), Phoxichilidium femoratum (Ballesteros et

al. 2021) and P. opilio (PRJNA236471); and the genomes of Pardosa

10 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

pseudoannulata (PRJNA512163), Acanthoscurria geniculata (PRJNA222716), M.

martensii (CDS sequence file; Cao et al. 2013). We then predicted the peptide

sequences using the Translate ExPASy online tool

(https://web.expasy.org/translate/; default settings). Protein sequence identity was

confirmed by reciprocal BLAST against the NCBI database and the construction of

maximum likelihood trees. Where more than one sequence was identified as a

potential candidate for a single gene, nucleotide and protein alignments were

inspected to eliminate the possibility that they were isoforms, individual variants, or

fragments of the same gene. Only the longest isoforms and gene fragments were

selected for phylogenetic analysis (Supplementary File 1). All Hox sequences

identified contain a complete or partial homeodomain (Supplementary Files 2-3). The

TBLASTN search in P, phalangioides identified a Ubx gene not reported previously

by Leite et al. (2018). The Trinity transcript accession numbers of all identified

sequences, NCBI protein accession and other sequence identifiers used in the

subsequent phylogenetic analysis are found in Supplementary File 1.

Due to high levels of fragmentation in P. opilio, multiple non-overlapping fragments

were found to align with query Wnt sequences. To verify the identity and relationship

of these fragments, primers were designed against the 5’-most and 3’-most ends of

the aligned series (see Supplementary File 1). Total RNA was extracted using Trizol

(Invitrogen) according to the manufacturer’s instructions and cDNA produced using

the SuperScriptII first-strand synthesis system (Invitrogen) for RT-PCR using the

designed primer pairs. PCR products were sequenced by Macrogen Europe to

confirm predicted combinations of fragmented transcripts (see Supplementary File

1).

Phylogenetic analysis

11 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Hox, Wnt and Frizzled protein predictions were retrieved from NCBI for the insects

Drosophila melanogaster, Bombyx mori, Tribolium castaneum; the crustacean

Daphnia pulex; and the onychophoran Euperipatoides kanangrensis (Supplementary

File 1). The Hox, Wnt and Frizzled peptide sequences for the myriapod Strigamia

maritima were retrieved from Chipman et al. (2014) (Supplementary File 1).

Alignments were performed in Clustal Omega using default parameters (Larkin et al.

2002; Sievers et al. 2011), with the exception of the full Hox protein sequences,

which were aligned using COBALT (Papadopoulos and Agarwala 2007). Maximum

likelihood trees were generated from whole-sequence alignments to assign

sequences to families and study the relationship between candidate duplicates.

Phylogenetic analyses were performed in IQ-Tree (v2.0.3, Nguyen et al. 2015) using

ModelFinder to identify optimal substitution models (JTT+F+R9 for full Hox

sequences, LG+G4 for Hox homeodomain sequences, GTR+F+I+G4 for Hox

homeobox sequences, LG+R8 for Wnt, JTT+R8 for Fz; Kalyaanamoorthy et al.

2017) and 100,000 bootstrap replicates. Trees were visualized in FigTree v.1.4.4

(http://tree.bio.ed.ac.uk/software/figtree/) and tidied in Adobe Illustrator. Hox

sequences were additionally analysed using RaXML v8 (Stamatakis 2014), using the

same substitution models chosen by Iqtree and the automatic bootstopping algorithm

(Pattengale et al. 2009). Alignments are provided in Supplementary Files 2-4 and 9-

11.

RESULTS

Transcriptome assemblies

12 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

To further study the outcomes of WGD in the ancestor of arachnopulmonates we

carried out RNA-Seq on embryos of two further spider species, P. amentata and M.

muscosa, and two species of amblypygids, C. acosta and E. bacillifer.

RNA-Seq for the four species produced between 222,479,664 and 272,844,971 raw

reads, reduced to 211,848,357 and 260,853,757 after processing. Trinity assembled

between 184,142 and 316,021 transcripts in up to 542,344 isoforms (Table 1).

Contig N50 ranged from 592 bp in M. muscosa to 978 bp in E. bacillifer, and from

1461 bp (M. muscosa) to 2671 bp (E. bacillifer) in the most highly expressed genes

(representing 90% of total normalized expression) (Table 1).

Transcriptomes were found to be between 83.7% (C. acosta) and 89.4% (E.

bacillifer) complete according to BUSCO scores compared to the arthropod

database, with between 3.5% and 9.5% duplicated BUSCOs. Compared to the

arachnid databases, transcriptomes were 82%-90.1% complete for single-copy

BUSCOs and contained between 5.3%-12.9% duplicated BUSCOs (Table 1).

To explore the extent of duplication in these arachnopulmonates we then surveyed

the copy number of Hox, Wnt and Frizzled genes in their transcriptomes in

comparison to other arachnids. It is important to note that the absence of genes

recovered from transcriptomes does not eliminate the possibility that they are

present in the genome, as the transcriptomes will only capture genes expressed at

the relevant point in development. Mixed-stage embryonic samples (all except E.

bacillifer) may yield more transcripts for the same reason.

Hox repertoires and their origins

13 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Spider Hox gene repertoires are largely consistent with Parasteatoda tepidariorum,

which has two copies of all except ftz (Figure 1). There are several exceptions:

single copies of Hox3 in Marpissa muscosa, Pardosa amentata and Pardosa

pseudoannulata, of Sex combs reduced (Scr) in Acanthoscurria geniculata and

Stegodyphus dumicola, of proboscipedia (pb) in A. geniculata, and of labial (lab) in

Pholcus phalangioides (Figure 1). Although we found two AbdB candidates in

Pardosa pseudoannulata, one of these did not contain a homeodomain and was

excluded from phylogenetic analyses. In contrast to Leite et al. (2018) we recovered

two copies of Ubx from P. phalangioides. The inclusion of additional sequences in

this analysis also helped resolve issues in gene homologies previously presented in

P. phalangioides by Leite et al. (2018).

Both amblypygids exhibit extensive duplication of Hox genes, with two copies

recovered for all except for pb in C. acosta and pb and Ubx in E. bacillifer (Figure 1).

The scorpions Mesobuthus martensii and Centruroides sculpturatus exhibited similar

Hox repertoires, with two copies recovered for all ten genes, except for a single copy

of Hox3 in C. sculpturatus.

Among the non-arachnopulmonate arachnids, I. scapularis and P. opilio exhibited no

duplication of any Hox genes, in line with previous genomic surveys (Leite et al.

2018; Gainett et al. 2021). In the mite Tetranychus urticae, we did not identify Hox3

and abdA candidates, consistent with Grbić et al. (2011) and Ontano et al. (2020).

However, our BLAST search did not recover the Antp-2 copy previously identified by

Grbić et al. (2011). The resolution of several sequences from T. urticae was variable;

for example, previously identified Tu-ftz-1, Tu-ftz-2, Tu-AbdB and Tu-Ubx sequences

14 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

(Grbić et al. 2011) did not resolve correctly in the full Hox tree (Figure 2) but did in

the homeodomain and homeobox trees (Supplementary Files 5-8).

We also surveyed two non-arachnid chelicerates, a pycnogonid Phoxichilidium

femoratum and the horseshoe crab Limulus polyphemus, the latter of which has

undergone multiple rounds of WGD independent of the arachnopulmonates (Kenny

et al. 2016; Nong et al. 2020). We recovered single copies of all Hox genes except

Ubx and abdA, which were not found, in Phoxichilidium femoratum, consistent with

Ballesteros et al. (2021; Figure 1). Limulus polyphemus returned multiple copies of

all Hox genes except ftz and Ubx, including three copies of lab, pb, Hox3, Scr and

AbdB, five potential copies of Dfd, and two copies of Antp and abdA (Figure 1).

Full protein sequences produced the best supported phylogeny: six nodes returned

support <50%. Two of these concerned the placement of outgroup (non-chelicerate)

sequences and one concerned the deeper relationship of the Hox3 and Pb clades,

which is beyond the scope of this study. The remaining three reflect uncertainty in

the within-clade placement of chelicerate sequences (Figure 2). An additional ten

nodes returned support of 50-60%, which we consider too weak to justify

interpretation. Analyses using protein sequences from homeodomains only

contained insufficient phylogenetic information to resolve within-clade relationships,

but confirmed gene identity (Supplementary Files 5-6). Nucleotide sequences of

homeodomains provided more within-clade resolution, but with lower support than

full protein sequences (Supplementary Files 7-8). Using full protein sequences, we

recover Antp as a paraphyletic group, albeit with low support (61%). The

homeodomain phylogenies (both protein and nucleotide) confirm the identity of these

sequences, indicating substantial sequence divergence outside of this conserved

15 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

region in several species. The following discussion is based on the phylogeny of the

full protein sequences (Figure 2).

Overall, the resolution of duplicate Hox sequences indicates that the majority are

likely to be ohnologs. In many cases, duplicates form well-supported clades of

orthologs, containing sequences from multiple Orders, suggesting a shared origin for

duplication. The majority of Hox genes are present in duplicate across the

arachnopulmonates, strengthening this pattern. Few duplicates form paralogous

pairs within species, or paired clades of paralogs within lineages, as would be

expected from lineage-specific duplications (although there are exceptions, such as

scorpion Lab and Antp, Figure 2).

For both abdA and AbdB, sequences broadly formed two overall clades; although

these were not well supported for AbdB (49-51%), relationships within them are still

informative. In both cases, spider sequences formed two distinct and well-supported

(>97%) clades that reflect overall phylogenetic relationships in their topology. The

amblypygid sequences resolved as two ortholog pairs, one in each of the two overall

clades. These are therefore strong candidates for ohnologs. Scorpion AbdB and

abdA were not consistent with this pattern; in both cases, one pair of orthologs

resolved together and one pair separately (Figure 2). Although this reflects a low

likelihood of a lineage-specific duplication of abdA and AbdB in scorpions, it does not

further clarify possible ohnolog relationships. Spider and scorpion Pb duplicates are

candidate ohnologs, resolving in two well-supported (>99%) clades of orthologs

whose topologies reflect phylogenetic relationships. Ftz duplicates in scorpions and

amblypygids also resolved with orthologs of other arachnopulmonates, but one set of

scorpion paralogs was placed with substantial uncertainty (support <60%). Most

spider and amblypygid Antp sequences follow the pattern expected of ohnologs,

16 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

forming two clades of orthologs, but three of the scorpion sequences formed a well-

supported clade (91%) while the fourth resolved within the small group of sequences

that fall outside the main Antp clade.

The origin of Ubx, Dfd, Lab, Hox3 and Scr duplicates in arachnopulmonates is not

clear from our phylogenetic analysis alone, with neither orthologs nor paralogs

resolving together consistently, and topology that is a poor reflection of phylogeny.

However, spider sequences broadly formed two clades with other

arachnopulmonates, and synteny analysis in both P. tepidariorum (Schwager et al.

2017) and Trichonephila antipodiana (Fan et al. 2021) demonstrate clearly that Hox

gene duplications therein are the result of WGD. The placement of the amblypygid

and scorpion duplicates was more variable. In some cases these also appear to

resolve as would be expected of ohnologs (e.g. amblypygid Hox3), but in others their

placement could indicate lineage-specific duplication, although usually with low

support (e.g. amblypygid Ubx).

It seems likely that having a single copy of ftz is common across all spiders (Figures

1-2), consistent with the loss of a duplicate in their common ancestor. A recently

published high-quality genome from Trichonephila antipodiana also reported a single

copy of ftz (Fan et al. 2021), as did another mygalomorph transcriptome,

Aphonopelma hentzi (Ontano et al. 2020). The absence of Hox3 duplicates in M.

muscosa, P. pseudoannulata and P. amentata may indicate a lineage-specific loss in

the RTA clade, which unites salticids, lycosids and their allies. Indeed, only one copy

of Hox3 was previously recovered in Cupiennius salei, which also belongs to the

RTA clade (Schwager et al. 2007). Although the absence of a second copy in the

embryonic transcriptomes could be attributed to failure to capture expression, both

C. salei and P. pseudoannulata yielded single copies from genomic DNA,

17 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

strengthening the case for a genuine loss. The P. tepidariorum Hox3-A sequence is

highly divergent, and its expression is very weak (Schwager et al. 2017); this could

indicate ongoing pseudogenisation. Other apparent losses of Hox gene duplicates

are restricted to transcriptomes of individual species, with the exception of Scr in

Stegodyphus dumicola, and could reflect failure to capture additional sequences.

The absence of a second copy of pb in both Charinus acosta and Euphrynichus

bacillifer, which are distantly related within Amblypygi, suggests a loss in the

common ancestor of amblypygids. Gainett and Sharma (2020) also recovered a

single copy of pb in Phrynus marginemaculatus, but two copies in Charinus

israelensis. However, one of these had an incomplete homeodomain (32 aa) that

was identical to the other C-isr protein sequence; we are hesitant about its status as

a duplicate. Embryos of C. acosta were collected at multiple stages of development,

supporting the hypothesis that this is a genuine loss, rather than absence of

expression at a particular developmental stage. However, the apparent absence of a

Ubx duplicate in E. bacillifer could equivocally indicate lineage-specific loss or

absence of expression at a single timepoint. In P. tepidariorum the two Ubx ohnologs

are expressed most strongly in close succession, at stages 8.1 and 8.2 (Schwager et

al. 2017), but this may not reflect relative expression patterns in amblypygids.

Wnt repertoires and their origins

Consistent with Parasteatoda tepidariorum (Janssen et al. 2010), we found

representatives of ten Wnt subfamilies in all surveyed spiders. The absence of Wnt9

and Wnt10 indicates their likely absence in the spider ancestor, while the absence of

Wnt3 is consistent with all other protostomes (Janssen et al. 2010; Murat et al. 2010;

Hogvall et al. 2014). Two copies of Wnt7 were retrieved from Marpissa muscosa,

18 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Pardosa amentata, Acanthoscurria geniculata, Pholcus phalangioides, P.

tepidariorum, Pardosa pseudoannulata and Stegodyphus dumicola. All spiders

except A. geniculata also yielded two copies of Wnt11. A second copy of Wnt4,

which is absent in P. tepidariorum, was recovered from P. amentata, M. muscosa, A.

geniculata, S. dumicola and P. pseudoannulata. We also found duplicates of

Wnt1/wg in both A. geniculata and P. pseudoannulata, and a duplicate of WntA in P.

pseudoannulata.

Representation of the Wnt subfamilies in the amblypygids is higher than any other

chelicerate studied to date, including those with high-quality genome assemblies

(Janssen et al. 2010; Hogvall et al. 2014; Holzem et al. 2019). We recovered

transcripts from twelve out of thirteen subfamilies (missing Wnt3) and duplicates of

Wnt1/wg, Wnt4, and Wnt7 in both species (Figure 3). Additional duplicates of Wnt6

and Wnt11 were recovered from Charinus acosta (Figure 3).

Representatives of ten Wnt subfamilies were found in the two scorpion genomes,

with both missing Wnt3, Wnt8 and Wnt9. Two copies of Wnt1/wg, Wnt6, Wnt7 and

Wnt11 were retrieved in both species, with an additional copy of Wnt4 in M.

martensii.

We found no evidence of Wnt gene duplication in the non-arachnopulmonate

arachnids (Figure 3). In the harvestman Phalangium opilio we recovered single

copies of all except Wnt2 and Wnt3. Ixodes scapularis was also missing Wnt10 but

was otherwise similar, whereas we also did not recover Wnt1/wg, Wnt7, Wnt9, or

Wnt11 in the mite Tetranychus urticae. In the non-arachnid chelicerates,

Phoxichilidium femoratum and Limulus polyphemus, we found similar representation

of the subfamilies, with all except Wnt3 and Wnt10 in P. femoratum and all except

19 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

these and Wnt9 in L. polyphemus. No duplicates were recovered in P. femoratum,

but large numbers of duplicates were found in L. polyphemus. These included six

potential copies of Wnt5 and Wnt7, four of Wnt11 and Wnt16, and five of WntA.

Only two nodes returned support <50%: one uniting Wnt8, Wnt9, Wnt10 and Wnt16

(39%), and one uniting Wnt1/wg, Wnt4, Wnt6 and Wnt11 (36%). These are

positioned deep within the tree and concern the interrelationships of the Wnt

subfamilies, which are beyond the scope of this study.

Most of the duplicate Wnt genes appear to be likely ohnologs; confirmation requires

synteny analysis, but the relationships between paralogs resolved by phylogenetic

analysis generally support duplications originating in the arachnopulmonate

ancestor.

Duplicates of Wnt7 were previously identified in P. tepidariorum (Janssen et al.

2010) and are recovered from all surveyed arachnopulmonates. The spider Wnt7

duplicates formed two clades (bootstrap ≥ 61%) suggesting the retention of ohnologs

(Figure 4). The amblypygid Wnt7 ortholog pairs resolve as sisters to the spider

clades, but support for these placements is lower (50-60%), and they display slightly

higher sequence similarity between paralogs (73-74%) than the spiders (60-72%).

The scorpion Wnt7 ortholog pairs formed their own separate clade with strong

support (92%; Figure 4), possibly indicating a lineage-specific duplication.

Nonetheless, the sequence divergence between the scorpion paralog pairs (68-70%)

is similar to that between putative ohnologs in spiders (60-72%).

Duplicates of Wnt11 previously identified in P. tepidariorum (Janssen et al. 2010)

were recovered from all surveyed spiders except A. geniculata, C. acosta, and both

scorpions. The spider Wnt11 duplicates formed two separate and well-supported

20 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

clades (≥ 85%, Figure 4), and each amblypygid Wnt11 orthology group was sister to

one of the spider clades (98-99%; Figure 4). Similarity between paralogs in the four

new transcriptomes was very low (41-54%). We propose that the spider and

amblypygid Wnt11 duplicates are probably retained from the ancestral WGD. The

resolution of the scorpion Wnt11 duplicates is less clear; ortholog pairs resolve

together with 100% support, but only one resolves as sister to the two clear clades

occupied by the spider and amblypygid duplicates (98%; Figure 4). They exhibit

similar sequence similarity (49-50%) to the putative ohnologs.

Wnt4 paralogs from M. muscosa, P. amentata, A. geniculata, P. pseudoannulata and

S. dumicola form two separate and well-supported clades with duplicates from the

amblypygids (bootstrap ≥ 91%). They show substantial sequence divergence within

species (53-64% similarity), indicating that they are again likely to represent retained

ohnologs following the arachnopulmonate WGD, despite being lost in the lineage to

P. tepidariorum.

We did not recover duplicates of Wnt2, Wnt5, Wnt8-10, or Wnt16 in any

arachnopulmonate lineage. This suggests their loss shortly after WGD in the

common ancestor of all arachnopulmonates. Two dissimilar overlapping partial

sequences of WntA were recovered from the genome of P. pseudoannulata, but

these resolve as sister to one another (Figure 4) and are located on the same

scaffold, indicating a lineage-specific tandem duplication. We therefore suggest that

the WntA duplicate resulting from WGD was also lost in the spider ancestor.

We identified two copies of Wnt1/wg in the transcriptomes of both amblypygids and

A. geniculata, and in the genomes of P. pseudoannulata, C. sculpturatus, and M.

martensii. To the best of our knowledge, this is the first duplication of Wnt1/wg

21 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

reported in any animal. This requires critical interpretation. We can eliminate the

possibility of individual variation in C. acosta, as embryos are produced by

parthenogenesis and are therefore clones, and in C. sculpturatus and S. dumicola,

as the sequences were recovered from a single individual’s genome (Supplementary

File 1). Sequence similarity between paralogs was low (55-73%) compared to

similarity between orthologs at the order level (e.g. 91% between M. muscosa and P.

amentata), and comparable to orthologs at the class level (e.g. 61% between P.

tepidariorum and Ixodes scapularis), reducing the likelihood that we are detecting

allelic variation within individuals. We also inspected nucleotide alignments and

found lower paralog sequence similarity than evident from amino acid sequences

(65-69%), indicating synonymous evolution. Although synteny analysis is required for

conclusive confirmation, our phylogeny indicates that the amblypygid, scorpion, and

A. geniculata duplicates are likely to be ohnologs retained from the

arachnopulmonate WGD, as they form separate clades (≥ 70%, Figure 4). The

putative duplicates in P. pseudoannulata, in contrast, resolve as sister to one

another. These two sequences have (short) non-identical overlapping regions, but

they are partial. It is equivocal whether they represent a genuine, lineage-specific

duplication.

Frizzled repertoires and their origins

All surveyed spiders possess at least one copy of FzI and FzII, with a second copy

recovered from the genome of Stegodyphus dumicola and the transcriptome of

Marpissa muscosa, respectively (Figure 5). FzIII was absent from the transcriptomes

of M. muscosa and P. amentata, and the genomes of S. dumicola and P.

pseudoannulata, consistent with P. tepidariorum (Janssen et al. 2015). However,

single FzIII orthologs were recovered from Acanthoscurria geniculata and previously

22 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

identified in Pholcus phalangioides (Janssen et al. 2015). Thus, entelegynes may

universally lack fz3 but it was likely present in the ancestor of all spiders. Consistent

with P. tepidariorum (Janssen et al. 2015), we identified two FzIV sequences in S.

dumicola and A. geniculata, but only one in M. muscosa, P. amentata and P.

pseudoannulata and only one was previously recovered from P. phalangioides

(Janssen et al. 2015). Janssen et al. (2015) demonstrated that the expression of the

two fz4 paralogs in P. tepidariorum is separated temporally, so we might not expect

to detect both transcripts in developing embryos of similar stages. However, this

does not explain the absence of a FzIV duplicate from the genome of P.

pseudoannulata.

Both amblypygid species have a large repertoire of frizzled genes compared to other

arachnids, with duplicates of FzI, FIII and FzIV in both species and an additional

duplicate of FzII in Charinus acosta (Figures 5).

The scorpions Mesobuthus martensii (Janssen et al. 2015) and Centruroides

sculpturatus possess single FzI and FzIII orthologs and a duplication of FzIV. We

also recovered a FzII ortholog in C. sculpturatus, and a FzI duplicate in M. martensii.

Among the non-arachnopulmonate arachnids, Ixodes scapularis possesses a single

copy of all four orthology groups, but FzIII was absent in Phalangium opilio and

Tetranychus urticae. A FzII duplicate was recovered from Tetranychus urticae.

Phoxichilidium femoratum and Limulus polyphemus possess all four orthology

groups. No duplicates were recovered in P. femoratum. Five copies of FzI and FzII

and three copies of FzIII and FzIV were recovered from L. polyphemus (Figure 5).

Frizzled paralogs appear to stem from both WGD events and lineage-specific

duplications. FzI duplicates were recovered from both amblypygid transcriptomes, M.

23 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

martensii, and S. dumicola. The Sd-Fz1 and Me-Fz1 paralog pairs exhibit high

sequence similarity (>73%) and resolved as sisters with 100% bootstrap support,

indicating that these are likely the results of lineage-specific duplications.

Conversely, the amblypygid Fz1-1 and Fz1-2 ortholog pairs form separate clades

with the Fz1 sequences from spiders and scorpions, respectively (Figure 6). We

therefore interpret these duplicates as ohnologs.

Duplicates of FzII were recovered from M. muscosa and C. acosta. The Mm-Fz2

paralogs form a well-supported clade (79%; Figure 6), indicating that this is the result

of a lineage-specific duplication followed by rapid sequence divergence in Mm-Fz2-2

(53% sequence similarity). The origin of the FzII duplication in Charinus acosta is not

clear. Ca-Fz2-1 resolves as a sister group to the spider Fz2 sequences (99%; Figure

6) and Ca-Fz2-2 forms a clade with Eb-Fz2, which in turn resolves as sister to the

Ca-Fz2-1 and spider sequences (93%; Figure 6). This topology could support an

ohnolog relationship between Ca-Fz2-1 and Ca-Fz2-2 but cannot be confirmed.

Sequence similarity between the C. acosta paralogs is relatively high (82%), perhaps

higher than expected from ohnologs.

The origin of the amblypygid FzIII duplicates is also unclear. One ortholog pair forms

a clade with L. polyphemus Fz3 (66%; Figure 6) and the other forms a clade with the

spiders and scorpions (≥ 70%; Figure 6). This suggests an origin in WGD, but

support for their placement is not strong (66%, Figure 6). Paralogous pairs

demonstrate middling sequence similarity (65-66%).

Both amblypygids and scorpions, and the spiders P. tepidariorum, S. dumicola and

A. geniculata, possess FzIV duplicates. The spider Fz4 sequences formed two

separate and well-supported clades (100%; Figure 6). The amblypygid ortholog pairs

24 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

resolved as sister to the two spider clades (98%; Figure 6). The scorpion ortholog

pairs, however, formed a clade together (76% support; Figure 6) which is sister to all

the spider and amblypygid Fz4 sequences (82%; Figure 6). All paralogous pairs

exhibited substantial sequence divergence (similarity 44-58%). We propose that the

spider and amblypygid FzIV duplicates are retained from the ancestral WGD;

however, once again, the origin of the scorpion FzIV duplicates is less clear, and

may reflect a lineage-specific duplication.

DISCUSSION

The impact of WGD on genome evolution is evident across the three gene families

surveyed here, but it appears that retention patterns of putative ohnologs vary

substantially between them. We also see distinct phylogenetic patterns beginning to

emerge in gene repertoires, with improved sampling within lineages enabling us to

distinguish between possible signal and likely noise. Of course, transcriptomic data

come with necessary caveats regarding gene expression and capture, and absences

from transcriptomes should be regarded with caution. However, where patterns are

consistent across multiple species, or better, between transcriptomic and genomic

resources, we can be more confident about their authenticity. For example,

comparisons of the transcriptome of Pardosa amentata and the genome of Pardosa

pseudoannulata show identical Hox and Fz repertoires, indicating good gene capture

in the former. The apparent duplicates of Wnt1/wg and WntA in P. pseudoannulata

that were not recovered in P. amentata are of uncertain status (partial sequences,

phylogenetic position) and were absent from all other spider genomes.

Hox duplicates are broadly retained, with slight variation between major clades

25 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

The widespread retention of duplicate Hox genes is consistent among the

arachnopulmonate orders studied to date, and specific repertoires appear to be fairly

conserved at the order level (Schwager et al. 2007; Cao et al. 2013; Di et al. 2015;

Schwager et al. 2017; Leite et al. 2018; Ontano et al. 2020). Given that this is the

level at which overall body plans are conserved, this is perhaps not surprising.

Thanks to the relatively conserved expression patterns of Hox genes along the

antero-posterior axis of chelicerates, we can begin to speculate about the possible

macroevolutionary implications of duplication and loss. For example, an anticipated

duplicate of pb appears to have been lost in both amblypygids but persists in spiders

and scorpions. In spiders, both pb paralogs are expressed in the pedipalp and leg-

bearing segments, separated temporally (Schwager et al. 2017). Given the highly

derived nature of the raptorial pedipalps and the antenniform first pair of walking legs

in amblypygids, it is perhaps surprising that this duplicate was not retained.

However, this might indicate that other Hox genes expressed in the anterior

prosomal segments (e.g. lab, Hox3, or Dfd) may contribute to these morphological

innovations. Recent work by Gainett and Sharma (Gainett and Sharma 2020)

examining the specification of the antenniform legs found little difference in Distal-

less, dachshund or homothorax expression between that and posterior leg pairs,

indicating that these are not likely to be responsible. A good candidate for future

study might be lab: a single ortholog is expressed in both the pedipalps and the first

walking leg in the harvestman Phalangium opilio (Sharma et al. 2012), and

expression patterns and experimental manipulation provide evidence for functional

divergence between the two lab paralogs, also expressed in the pedipalps and first

walking legs, in P. tepidariorum (Pechmann et al. 2015; Schomburg et al. 2020).

26 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Likewise, the seemingly universal absence of a ftz duplicate in spiders, but its

retention in scorpions and amblypygids, poses a similar question. In both P. opilio

(Sharma et al. 2012) and P. tepidariorum (Schwager et al. 2017), a single copy of ftz

is expressed in leg pairs 2-4, implying that spiders have not lost ftz functionality by

losing a duplicate. Any subfunctionalization or neofunctionalization that could be

evident in scorpion and amblypygid ftz had therefore presumably not taken place at

the point of their divergence from spiders. Expression patterns of the two paralogs in

these groups would be of interest for comparison with both harvestmen and spiders.

Given that neither scorpions nor amblypygids exhibit much divergence within L2-4,

there are no obvious routes for subfunctionalization.

The potential loss of a Hox3 duplicate in the spider RTA clade would be an unusual

example of infraorder variation in Hox repertoires, if it represents a true absence.

The consistency of this finding between both transcriptomic and genomic resources

supports this conclusion. In P. tepidariorum, Hox3-A and Hox3-B exhibit dramatic

protein sequence divergence (20.6% similarity). Both copies are expressed in the

embryo, with their expression overlapping spatially and temporally but not identical,

indicating some functional divergence; however, Hox3-A expression was reported to

be very weak (Schwager et al. 2017). In the other spiders for which we recovered

Hox3 duplicates, paralogs also exhibited very low sequence similarities (24.7-

30.6%), and these duplicates had very low similarity to each other (17.2-30.1%,

compared to 33-58.2% between those resolving with other spider Hox3 sequences).

In all cases, one resolved within a well-supported clade of spider Hox3 and the other

was placed haphazardly. We speculate that the Hox3 duplicate is highly divergent or

degenerate in spiders, leading to eventual pseudogenization in the RTA clade. As

other infraorder losses of Hox genes were only observed in single species, some

27 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

from embryonic transcriptomes, it would be premature to conclude that they are

genuinely absent from the genome.

The retention of Wnt duplicates is more specific, but with some surprises

In contrast to the widespread retention of Hox duplicates, these new data indicate

that the retention of duplicate Wnt genes is less common and restricted to certain

subfamilies. Apparent ohnologs of Wnt4, Wnt7 and Wnt11 are retained in the

majority of arachnopulmonates, for example, but Wnt2, Wnt5, Wnt8-10, and Wnt16

are present in single copies across the board. These patterns could reflect early

losses, by chance, of duplicates, or differential ‘evolvability’ of Wnt subfamilies. The

fact that retention patterns appear to differ between the arachnopulmonate and

horseshoe crab independent WGD events lends support to the former hypothesis.

Our understanding of specific Wnt functions among arthropods is more limited than

that of Hox genes, but Wnt expression patterns in Parasteatoda tepidariorum are

available for tentative comparison. For example, previous attempts to characterise

the expression patterns of Wnt11 paralogs in P. tepidariorum only detected

expression of Wnt11-2 (Janssen et al. 2010). Given the retention of Wnt11-1 in both

spiders and scorpions, and the considerable divergence between paralogous

sequences, Wnt11 could be a good candidate for sub- or neofunctionalization, but

the role of Wnt11-1 remains unknown. Conversely, the presence of two apparent

Wnt4 ohnologs in spiders and amblypygids (Figures 3-4) contrasts with the retention

of only one Wnt4 in P. tepidariorum. The role of the additional copy, particularly in

spiders, will be of future interest and expression patterns of Wnt4-2 in these groups

will help to clarify this. Although it is possible that the second copy is redundant or in

the process of pseudogenization, the fact that both ohnologs are retained in two

28 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

large clades, with detectable levels of expression during development, suggests that

this is not the case. Thus, its absence in P. tepidariorum unexpectedly appears to be

the exception.

The discovery of duplicate Wnt1/wg in scorpions, amblypygids and mygalomorphs is

particularly exciting: duplicates of this Wnt gene have not yet been detected in any

other metazoans, even following multiple rounds of WGD in vertebrates, teleosts

(see https://web.stanford.edu/group/nusselab/cgi-bin/wnt/vertebrate), or horseshoe

crabs. Wnt1/wg performs a wide variety of roles in arthropods, including in segment

polarization and in appendage and nervous system development (Murat et al. 2010)

and has an accordingly complex expression pattern in P. tepidariorum, appearing in

the L1 and L2 segments, limb buds, and dorsal O2 and O3 segments (Janssen et al.

2010). In theory, therefore, there is ample potential for subfunctionalization. Gene

expression and functional studies of Wnt1/wg duplicates in arachnopulmonates will

no doubt prove extremely interesting in the future.

The presence of Wnt10 in both amblypygids and Phalangium opilio is also intriguing

because it is absent from all other chelicerates surveyed so far. Whether this

indicates multiple losses of Wnt10 in all other arachnid lineages, the recovery of a

lost Wnt10 in amblypygids and harvestmen, or the co-option of another gene, is

unclear. It is notable, however, that Wnt10 is also absent in both Limulus

polyphemus and Phoxichilidium femoratum, non-arachnid chelicerates. This might

favour the recovery or co-option hypotheses.

Fz repertoires vary substantially

Previous studies of spiders, scorpions, and ticks indicated that Frizzled repertoires in

these groups are restricted to three or four copies, often with incomplete

29 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

representation of the four orthology groups (Janssen et al. 2015). The spiders

Marpissa muscosa, Stegodyphus dumicola, Pardosa pseudoannulata and Pardosa

amentata are consistent with this pattern, albeit with a unique duplication of FzII in

the jumping spider. We also recovered a single copy of FzII in Centruroides

sculpturatus, which is absent in Mesobuthus martensii, and a FzI duplicate in M.

martensii that was previously missed. In contrast, all four frizzled subfamilies were

recovered in both amblypygid species, with three present in duplicate in

Euphrynichus bacillifer and four in Charinus acosta. Based on our data, it appears

that the frizzled repertoire of amblypygids is around twice the size of all other

arachnids and may have followed a very different evolutionary trajectory to spiders

and scorpions following WGD. The expanded repertoire of frizzled genes in

amblypygids is intriguing since they have the widest Wnt repertoires, via both

duplication and representation of the subfamilies (Wu and Nusse 2002). However,

although frizzled genes encode key receptors for Wnt ligands, they have other Wnt-

independent functions, so the expansion of the frizzled gene repertoire could be

equally related to the evolution of alternative signalling roles (Janssen et al. 2015; Yu

et al. 2020).

Arachnopulmonate genome evolution in the wake of WGD

Our new analyses provide a thorough survey of Hox, Wnt and frizzled genes in

arachnids, and substantially improve the density and breadth of taxonomic sampling

for these key developmental genes in Arachnopulmonata. We find evidence of

consistent evolutionary trajectories in Hox and Wnt gene repertoires across three of

the five arachnopulmonate orders, with inter-order variation in the retention of

specific paralogs. We have also identified intraorder variation at the level of major

clades in spiders, which could help us better understand their morphological

30 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

evolution. In new data for a third arachnopulmonate lineage, the amblypygids, we

find additional evidence supporting an ancestral WGD and are better able to

reconstruct the chronology of gene duplications and losses in spiders and scorpions.

These transcriptomic resources are among the first available for amblypygids and

will aid future investigations of this fascinating group.

By improving taxonomic coverage within the spider lineage we are better able to

polarise some loss/duplication events and identify potential new trends within the

spiders, particularly illustrating separations between synspermiatan and entelegyne

spiders, and between the derived RTA clade and other spiders. Despite being

unable to ultimately conclude that some missing transcripts reflect genuine genomic

losses, it appears that the evolution of these developmental genes in spiders is more

complicated than we thought. It may be that these gene repertoires are genuinely

more variable within spiders than they are in amblypygids or scorpions; spiders are

by far the most taxonomically diverse arachnopulmonate order, and the apparent

diversity of repertoires may simply reflect this. Conversely, the higher apparent

intraorder diversity of gene repertoires may be an artefact of increased sampling in

spiders (up to four or five species for specific gene families) compared to the one or

two available resources for scorpions and amblypygids; we may detect more

diversity within these groups with increased sampling. Nonetheless, we see two

notable trends within spiders, outlined below.

First, we see several characters that appear to unite the RTA clade, which contains

almost half of all extant spider species (World Spider Catalog 2019), having

diversified rapidly following its divergence from the orb weavers (Garrison et al.

2016; Fernández et al. 2018; Shao and Li 2018). Marpissa muscosa, Pardosa

amentata, and Pardosa pseudoannulata all exhibit the apparent loss of Hox3 and fz4

31 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

paralogs and the retention of a Wnt4 duplicate. The identification of genetic trends

potentially uniting this group is exciting, even if the macroevolutionary implications

are unclear: as described above, the possible functions of a Wnt4 paralog are

elusive. Members of the RTA clade are very derived compared to other

araneomorph spiders, both morphologically (e.g. male pedipalp morphology and

sophisticated eyes) and ecologically (most are wandering hunters), and their rapid

diversification would align with clade-specific genetic divergence (Garrison et al.

2016; Fernández et al. 2018; Shao and Li 2018).

Second, although data are only available for single representatives of the

plesiomorphic clades Synspermiata (Pholcus phalangioides) and Mygalomorphae

(Acanthoscurria geniculata), these hint at lineage-specific losses of Hox paralogs

and recover the only examples of FzIII found in spiders so far. The presence of FzIII

is consistent with other arachnopulmonate groups and suggests that it was present

in the spider ancestor and only lost in the more derived entelegyne lineages. If

selected Hox duplicates are indeed absent from the genomes of these two species,

this represents an interesting divergence between the three major groups of spiders.

Although it may seem unusual for Hox duplicates to be lost within lineages, a recent

high-quality genome of Trichonephila antipodiana revealed likely absences of Hox3,

Ubx and abdA ohnologs in addition to ftz (Fan et al. 2021). Though they are unlikely

to be directly responsible, the apparent divergence in gene repertoires we see

between A. geniculata, P. phalangioides and the other spider lineages might provide

a starting point for understanding the important morphological differences between

mygalomorphs, synspermiatans and entelegynes. However, genomic information for

additional taxa in both groups is required to verify these potential losses.

32 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

The amblypygids emerge as a key group of interest for studying the impacts of WGD

owing to their high levels of ohnolog retention. Our transcriptomes, from

representatives of two major clades, provide new evidence supporting a WGD in the

ancestor of arachnopulmonates and demonstrating widespread retention of ohnologs

in three major families of developmental genes (consistent with the retention of many

duplicated regulators of eye development in other species, Gainett et al. 2020). In all

three gene families we studied, repertoires were largest in the amblypygid species.

This was particularly the case in Charinus acosta, which belongs to the less

speciose and more plesiomorphic infraorder Charinidae within living Amblypygi.

Although this study represents just two amblypygid species and three gene families,

this appears to contradict widespread predictions of diversification with the

duplication of important developmental genes such as Hox (e.g. Van De Peer et al.

2009). Of particular interest are the amblypygid Wnt gene repertoires. We have

identified from their transcriptomes, and from the published genome of Centruroides

sculpturatus, the first reported duplicates of Wnt1/wg in any animal, as well as the

first reported Wnt10 in any arachnid. Future functional studies of these genes and

their expression during development will be critical to understanding the evolutionary

impacts of these unusual components of amblypygid gene repertoires. Amblypygids

also represent a potential model group for studying the evolution of arthropod body

plans, owing to the unusual and derived morphology of the pedipalps and especially

the first walking legs. Thanks to a substantial existing body of work on anterior-

posterior patterning, segmentation and appendage development in spiders and other

arachnids, we may have a chance to crack the genetic underpinnings of these

dramatic evolutionary innovations (Pechmann et al. 2009; Sharma et al. 2012;

33 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Sharma et al. 2014; Turetzek et al. 2016; Schwager et al. 2017; Turetzek et al. 2017;

Schomburg et al. 2020; Baudouin-Gonzalez et al. 2021) .

Finally, our analysis of existing genomic data for Centruroides sculpturatus and

Mesobuthus martensii has recovered several Wnt and Frizzled gene duplications,

similar to spiders and amblypygids. However, in contrast to those groups, our

phylogenies have sometimes supported within-lineage duplication in scorpions, as

opposed to the retention of ohnologs following WGD, even when these are observed

in spiders and amblypygids. This was the case for AbdB, Wnt1/wg, Wnt6, Wnt7, and

FzIV (Figures 2,4,6). However, levels of sequence similarity in these cases were

comparable for C. sculpturatus paralogs and amblypygid and spider ohnologs, when

we might expect within-lineage duplicates to show higher similarity. The resolution of

the paralogous sequences in our phylogenetic analyses could be confounded by the

early-branching position of scorpions within Arachnopulmonata, which means

paralogs would be expected to appear towards the bottom of ortholog clades and are

more vulnerable to movement. Nonetheless, this pattern emerged multiple times in

our analyses and may be of future interest.

Both arachnopulmonates and horseshoe crabs have been subject to WGD.

Comparison between these independent events is a useful tool in studying WGD,

spanning smaller phylogenetic distances than arachnopulmonate-vertebrate

comparisons. From the three gene families surveyed here, both patterns and

inconsistencies emerge. As in arachnopulmonates, Hox gene duplicates appear to

be overwhelmingly retained, even in triplet or quadruplet in some cases, in Limulus

polyphemus. Ftz is an interesting exception that aligns with the absence of

duplicates in spiders, but not scorpions or amblypygids. However, the apparent loss

of all Ubx and two abdA duplicates stands at odds with the arachnopulmonates,

34 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

wherein these are largely retained. Wnt repertoires follow a similar pattern to

arachnopulmonates - only select genes are retained in duplicate - but its application

is very different: for example, whereas Wnt4 duplicates are common in

arachnopulmonates, they are completely absent in L. polyphemus, and vice versa for

Wnt5. There are, however, some Wnts that are commonly or even universally

present in single copies following both independent WGD events, potentially

indicating low potential for sub- or neofunctionalization, such as Wnt8, Wnt6 and

Wnt1/wg.

Overall, our new data provide further evidence of an ancestral arachnopulmonate

WGD, identify evolutionary patterns within gene families following WGD, reveal new

diversity in spider gene repertoires, better contextualise existing data from spiders

and scorpions, and broaden the phylogenetic scope of available data for future

researchers. However, other arachnid groups, both with and without ancestral WGD,

require further study. Recent work on two pseudoscorpions recovered duplicates of

most Hox genes (Ontano et al. 2020), in contrast to previous surveys (Leite et al.

2018). This not only further supports arachnopulmonate WGD but substantially

improves our understanding of pseudoscorpion placement within arachnids, which

has been historically problematic. The sequencing of a pseudoscorpion genome

provides the tantalising chance to add a fourth lineage to future studies of WGD and

its impacts (Ontano et al. 2020). The remaining arachnopulmonate orders,

thlyphonids (vinegaroons or whip scorpions) and schizomids, form a clade with

amblypygids (Pedipalpi) and should also have been subject to the

arachnopulmonate WGD. Future work on these groups will shed light on the unusual

patterns of gene retention we find in both major clades of amblypygids. Non-

arachnopulmonate arachnids are invaluable for contextualising the changes that

35 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

occur following WGD, both in terms of gene repertoires and of gene function.

Genomic resources and gene expression pattern studies are vital for this, and the

harvestman Phalangium opilio has emerged as the clear model species (Sharma et

al. 2012; Gainett et al. 2021). The ability to compare rates of sequence divergence,

within-lineage gene duplication, and, eventually, functional properties of

developmental genes in these groups will provide critical comparative data for

arachnopulmonates.

Availability of data and materials

Alignments and gene accession numbers are included as supplementary information

files. Transcriptomes are available via SRA (BioProject PRJNA707377). Assemblies

will be made publicly available upon acceptance for publication.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the John Fell Fund, University of Oxford (award

0005632 to LSR), NERC (NE/T006854/1 to APM and LSR), the Leverhulme Trust

(RPG-2016-234 and RPG-2020-237 to APM and LSR, respectively), and the

Biotechnology and Biological Sciences Research Council (BBSRC) (grant number

BB/M011224/1). These funding bodies were not involved in the design of this study,

data collection or analysis, or in writing the manuscript.

Authors’ contributions

36 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

AH analysed assembled transcriptomes, identified gene candidates, performed

phylogenetic analyses, interpreted data, prepared figures and wrote sections of the

manuscript. LBG contributed to data analysis and interpretation. AS processed

embryos and performed RNA extractions. RJ provided data for Pholcus

phalangioides and Phalangium opilio. MS provided amblypygid embryos. MH

provided sequence data and contributed to phylogenetic analysis. SA assembled

transcriptomes, analysed assembly quality and wrote sections of the manuscript.

APM designed and supervised the project, contributed to data interpretation and

analysis, and wrote sections of the manuscript. LSR designed and supervised the

project, performed phylogenetic analyses, interpreted data, prepared figures and

wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors are very grateful to Philip Steinhoff and Gabriele Uhl (University of

Greifswald) for providing embryos of Marpissa muscosa, to Matthias Pechmann

(University of Köln), Natascha Turetzek (Ludwig-Maximilians-University of Munich)

and Prashant Sharma (University of Wisconsin Madison) for providing assembled

transcriptomes of Acanthoscurria geniculata, Pholcus phalangioides and Phalangium

opilio, respectively, to Nathan Kenny for helpful comments on the manuscript, and to

Simon Ellis (University of Oxford) for IT support.

37 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

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49 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Figure 1 Repertoires of Hox genes in arachnids and other selected arthropods.

Hox genes are represented by coloured boxes with duplicated Hox genes indicated

by overlapping boxes. Figure includes Hox repertoires previously surveyed in the

arachnids P. tepidariorum, Centruroides sculpturatus, Mesobuthus martensii,

Phalangium opilio, I. scapularis (all genomes) and Pholcus phalangioides (embryonic

transcriptome), the myriapod Strigamia maritima and the insects Drosophila

melanogaster, Tribolium castaneum and Bombyx mori. The insect Hox3 homolog

zen has undergone independent tandem duplications in T. castaneum to yield zen

and zen2; in cyclorrhaphan flies to yield zen and bicoid; and in the genus Drosophila

to yield zen2. Bombyx mori is not representative of all species of ditrysian

Lepidoptera, which typically possess four distinct Hox3 genes termed Special

homeobox genes (ShxA, ShxB, ShxC and ShxD) and the canonical zen gene.

Figure 2 Maximum likelihood phylogeny of Hox amino acid sequences. The

Hox genes are shown as different colours (after Figure 1). Panarthropods included:

Acanthoscurria geniculata (Ag), Bombyx mori (Bm), Centruroides sculpturatus (Cs),

Charinus acosta (Ca), Drosophila melanogaster (Dm), Euperipatoides kanangrensis

(Ek), Euphrynichus bacillifer (Eb), Ixodes scapularis (Is), Limulus polyphemus (Lp),

Marpissa muscosa (Mm), Mesobuthus martensii (Me), Parasteatoda tepidariorum

(Pt), Pardosa amentata (Pa), Pardosa pseudoannulata (Pan), Phalangium opilio

(Po), Pholcus phalangioides (Pp), Phoxichildium femoratum (Pf), Stegodyphus

dumicola (Sd), Strigamia maritima (Sm), Tetranychus urticae (Tu), and Tribolium

castaneum (Tc). Node labels indicate ultrafast bootstrap support values. See

Supplementary File 1 for accession numbers, Supplementary File 2 for full amino

acid sequence alignments.

50 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Figure 3 Repertoires of Wnt subfamilies in arthropods and an onychophoran.

The Wnt subfamilies (1-11, 16 and A) are represented by coloured boxes with

duplicated genes represented by overlapping boxes and putatively lost subfamilies

indicated by white boxes. Figure includes Wnt repertoires recovered in this study and

previously surveyed in the arachnids Parasteatoda tepidariorum and Ixodes

scapularis; the insects Drosophila melanogaster, Tribolium castaneum and Bombyx

mori; the crustacean Daphnia pulex; the myriapod Strigamia maritima; and the

onychophoran Euperpatoides kanangrensis.

Figure 4. Maximum likelihood phylogeny of Wnt amino acid sequences. The 12

Wnt subfamilies are shown as different colours (after Figure 3). Panarthropods

included: Acanthoscurria geniculata (Ag), Bombyx mori (Bm), Centruroides

sculpturatus (Cs), Charinus acosta (Ca), Drosophila melanogaster (Dm),

Euperipatoides kanangrensis (Ek), Euphrynichus bacillifer (Eb), Ixodes scapularis

(Is), Limulus polyphemus (Lp), Marpissa muscosa (Mm), Mesobuthus martensii

(Me), Parasteatoda tepidariorum (Pt), Pardosa amentata (Pa), Pardosa

pseudoannulata (Pan), Phalangium opilio (Po), Pholcus phalangioides (Pp),

Phoxichildium femoratum (Pf), Stegodyphus dumicola (Sd), Strigamia maritima (Sm),

Tetranychus urticae (Tu), and Tribolium castaneum (Tc). Node labels indicate

ultrafast bootstrap support values. See Supplementary File 1 for accession numbers,

Supplementary File 9 for amino acid sequence alignments, and Supplementary File

10 for nucleotide sequence alignments of Wnt1/wg duplicates in C. acosta, C.

sculpturatus and Euphrynichus bacillifer.

Figure 5 Repertoire of frizzled genes in arachnids and other selected

arthropods. The four frizzled orthology groups (FzI, FzII, FzIII, and FzIV) are

represented by coloured boxes, with duplicated genes represented by overlapping

51 Harper et al. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

boxes and gene loss represented by a white box. Figure includes frizzled repertoires

previously surveyed in the arachnids Parasteatoda tepidariorum, Mesobuthus

martensii, Ixodes scapularis (all genomes), and Pholcus phalangioides (embryonic

transcriptome); the myriapod Strigamia maritima; the insects Drosophila

melanogaster and Tribolium castaneum; and the onychophoran Euperipatoides

kanangrensis.

Figure 6 Maximum likelihood phylogeny of Frizzled proteins. The frizzled genes

are shown as different colours (after Figure 5). Panarthropods included:

Acanthoscurria geniculata (Ag), Bombyx mori (Bm), Centruroides sculpturatus (Cs),

Charinus acosta (Ca), Drosophila melanogaster (Dm), Euperipatoides kanangrensis

(Ek), Euphrynichus bacillifer (Eb), Ixodes scapularis (Is), Limulus polyphemus (Lp),

Marpissa muscosa (Mm), Mesobuthus martensii (Me), Parasteatoda tepidariorum

(Pt), Pardosa amentata (Pa), Pardosa pseudoannulata (Pan), Phalangium opilio

(Po), Pholcus phalangioides (Pp), Phoxichildium femoratum (Pf), Stegodyphus

dumicola (Sd), Strigamia maritima (Sm), Tetranychus urticae (Tu), and Tribolium

castaneum (Tc). Node labels indicate ultrafast bootstrap support values. See

Supplementary File1 for accession numbers and Supplementary File 11 for

alignments

52 Harper et al. bioRxiv preprint

Table 1. Assembly metrics for transcriptomes of Charinus acosta, Euphrynichus bacillifer, Marpissa muscosa and was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. doi:

Pardosa amentata. https://doi.org/10.1101/2020.07.10.177725

* Based on longest isoform per gene

** Same as contig N50 but based on top most highly expressed genes that represent 90% of the total normalized expression data

*** The number of genes for which Ex90N50 is calculated

1 10 species, n=2934 BUSCOs; C=Complete [D=Duplicated], F=Fragmented, M=Missing.

2 90 species, n=1013 BUSCOs; C=Complete [D=Duplicated], F=Fragmented, M=Missing. ; this versionpostedApril26,2021.

Processed #Trinity #Trinity Contig N50 Ex90N50 #Ex90N50 Arachnid BUSCO Arthropod BUSCO Species Raw Reads Reads Genes Isoforms (bases)* (bases)** genes*** Scores (C[D],F,M)1 Scores (C[D],F,M)2 C. acosta 272,844,971 260,853,757 237,678 334,267 896 2406 31,012 94.9%[12.9%],1.0%,4.1% 93.2%[9.5%],1.5%,5.3%,

E. bacillifer 249,938,618 239,034,000 184,142 285,861 978 2671 22,647 93.8%[7.1%],1.5%,4.7% 92.9%[3.5%],0.7%,6.4%

P. amentata 266,764,548 256,911,378 316,021 542,344 652 1758 38,423 95.4%[5.3%],0.9%,3.7% 92.9%[4.5%],1.2%,5.9%

M. muscosa 222,479,664 211,848,357 276,943 473,878 592 1461 46,196 94.7%[6.1%],1.3%,4.0% 90.8%[3.9%],1.9%,7.3% The copyrightholderforthispreprint(which

53 Harper et al. lab pb Hox3 Dfd Scr ftz AntpUbx abdAAbdB bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725Marpissa; this version muscosa posted April 26, 2021. 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. RTA WGD Pardosa amentata

Pardosa pseudoannulata

Entelegynae Stegodyphus dumicola

Parasteatoda tepidariorum Araneae Synspermiata Pholcus phalangioides

Mygalomorphae Acanthoscurria geniculata Arachnopulmonata Euphrynichus bacillifer Amblypygi

Charinus acosta

Arachnida Mesobuthus martensii Scorpiones Centruroides sculpturatus Eucherlicerata Opiliones

Cherlicerata Phalangium opilio

Parasitiformes Ixodes scapularis

Arthropoda Acariformes Tetranychus urticae Xiphosura Limulus polyphemus 5 Pycnogonida Phoxichilidium femoratum bcd zen Diptera Drosophila melanogaster Shx Lepidoptera Insecta Bombyx mori 16 Coleoptera Tribolium castaneum Mandibulata Crustacea Daphnia pulex

Myriapoda Strigamia maritima

Onychophora Euperipatoides kanangrensis Dm_NP_001303472.1_AbdB

Bm_XP_012549559.1_AbdB

Tc_NP_001034519.1_AbdB

92

90 Dp_EFX86798.1_AbdB

99 AbdB

Antp Pf_TRINITY_DN25812_c0_g1_i1_AbdB 98

Pt_XP_015911312.1_Hox3-a

Pan_SBLA01092602.1-1228036-1228266_AbdB_partial

98 Pa_TRINITY_DN10063_c0_g1_i9_AbdBMm_TRINITY_DN71971_c0_g1_i3_AbdB

98 Is_EEC15619.1_AbdB

Mm_TRINITY_DN34604_c0_g1_i1_Antp

Sd_XM_035362693.1_AbdB Pa_TRINITY_DN6307_c0_g1_i2_Antp 97 Ag_TRINITY_DN75690_c2_g1_i1_Antp Pp_c97115_g1_i1_AbdB

100 Pt_XP_015930562.1_AbdB_b Po_CCH51009.1_AbdB_partial 94

100

Pp_c103414_g2_i2_Antp Pt_NP_001310750.1_Antp-a Eb_TRINITY_DN4365_c0_g1_i1_Antp Po_CCH51006.1_Antp_partial Ca_TRINITY_DN3152_c0_g1_i6_Antp Ca_TRINITY_DN1632_c2_g1_i9_AbdB Sd_XM_035371268.1_Antp Eb_TRINITY_DN8255_c0_g1_i1_AbdB Lp_XP_022236342.1_AbdB Lp_XP_022245288.1_AbdB Mm_TRINITY_DN17582_c0_g1_i5_Antp Lp_XP_022237377.1_AbdB Cs_XP_023230594.1_Antp-a sI 100

_ 100 Ek_CCK73379.1_AbdB 93 Me_MMa29074_AbdB _PX_mB 97 Pa_TRINITY_DN5299_c0_g1_i7_Antp 100 100 Me_MMa46051_Antp-2 Tu_tetur20g02440_Antp-2

Lp_XP_022237372.1_Antp

Ca_TRINITY_DN20061_c0_g1_i1_Antp 100 Eb_TRINITY_DN662_c2_g1_i2_Antp Sm_SMAR004172_AbdB Pp_c105467_g2_i4_AntpPt_XP_015917912.1_Antp-bSd_XM_035363059.1_AntpCs_XP_023234920.1_Antp-b 20_PX

489 Cs_XP_023221400.1_AbdB-b

310_PX_pL 82

rc 77 89 Ca_TRINITY_DN45919_c0_g1_i1_AbdB 852 49 _ 1.5 6 5 9 4 5 2 1 0 Eb_TRINITY_DN15836_c0_g1_i1_AbdB 68 S_1 .1 Tc_NP_001034505.1_ptl

tnA

.37337KCC 100 p

100 100 94 _1.1276

100 ptnA_2

100 70 96

ptnA

100

001

ptn 100 100 Pa_TRINITY_DN7867_c0_g1_i1_AbdB 99 100 94 54 _k A_4614 Mm_TRINITY_DN7491_c0_g1_i1_AbdB 100 97

100 p

E 91 tnA_1.408 94 60 84 51 Pt_XP_015930590.1_AbdB_a 100 100 77 100

9

0 Sd_XM_035353428.1_AbdBPp_c106591_g2_i1_AbdB

8

0R 100 99

AM

Ek_CCK73375.2_Antp

68XFE_pD 97 Ag_TRINITY_DN77332_c0_g2_i1_AbdB

Dp_EFX86805.1_ftz S_

Tu_XP_015790206.1_Antp-1 100

mS

001 Cs_XP_023228868.1_AbdB-a 94 Scr Dm_NP_996167.1_Antp Me_MMa42819_AbdB

96

94

Mm_TRINITY_DN13983_c0_g1_i2_Scr

94 Pa_TRINITY_DN3878_c0_g2_i1_Scr Eb_TRINITY_DN4365_c0_g2_i1_Scr Ca_TRINITY_DN11320_c2_g1_i3_Scr

Eb_TRINITY_DN888_c2_g1_i1_ScrPt_XP_015921994.1_Scr-aCs_XP_023230598.1_Scr-a Dm_NP_001368995.1_Scr Pp_c109150_g1_i2_Scr

Me_MMa55596_Scr Cs_XP_023234859.1_Scr-b Dp_EFX86806.1_Scr Lp_XP_022244131.1_Scr Ag_TRINITY_DN103185_c0_g1_i1_Scr_partialCa_TRINITY_DN776_c4_g1_i1_ScrBm_NP_001037339.1_Scr Lp_XP_013792672.2_Scr Po_comp168984_c0_seq1_Scr_partialTc_NP_001034523.1_Cx Mm_TRINITY_DN3338_c1_g1_i3_Scr Pf_TRINITY_DN7017_c0_g1_i1_Scr Lp_XP_013794100.2_Scr Pan_SBLA01129491.1-734218-734517_Scr_partial Pa_TRINITY_DN3156_c0_g1_i4_ScrIs_XP_002406405.2_Scr

Me_MMa52910_Scr Pt_XP_021004339.1_Scr-bSd_XM_035368749.1_Scr 7

8 Pan_SBLA01591915.1-c93378-93118_Scr_partial 26

Tu_XP_015790203.1_Scr Sm_Scr 93 Pp_c101195_g2_i1_Scr 100 100

68 98 100 89 100 98 100 98 Dm_NP_477201.1_Dfd 92 85 39 80 96 64 53 Pan_SBLA01293003.1-c769617-769336_Antp_partial93 61 95 98 5165 46 51 Po_CCH51003.1_Dfd_partial Pan_SBLA01416513.1-c2731412-2731164_Antp_partialAg_TRINITY_DN43429_c0_g1_i1_Antp_partial93 57 Me_MMa42999_Antp-1 87 48 92 99 88 Tc_NP_001034510.1_Dfd 93 Pan_SBLA01529937.1-766703-767212_Dfd_partialBm_NP_001037341.1_Dfd Pa_TRINITY_DN17053_c0_g1_i7_Dfd 90 97 Ag_TRINITY_DN78131_c2_g1_i1_Dfd Pf_TRINITY_DN24197_c0_g1_i1_Antp 74 100 94 Tu_XP_015790154.1_AbdB Cs_XP_023235027.1_Ubx-a100 80 93 Ek_CCK73376.1_Ubx Is_XP_029842579.1_Ubx Tu_XP_015790291.1_Ubx100 Sd_XM_035352116.1_Dfd91 Dfd 100 Dp_EFX86808.1_Dfd Lp_XP_022237375.1_Ubx Pp_c96808_g3_i2_Dfd Pan_SBLA01093126.1-57593-58009_Dfd_partial 96 91 100Pa_TRINITY_DN1357_c1_g1_i4_Dfd Ag_TRINITY_DN59883_c0_g1_i1_UbxPp_c111834_g6_i1_Ubx 100 Mm_TRINITY_DN3338_c0_g1_i3_Dfd 93 Pt_NP_001310749.1_Dfd-a 98 83 100 100Sd_XM_035377715.1_Dfd Sd_XM_035349894.1_Ubx 89 Ag_TRINITY_DN54692_c0_g1_i1_Dfd 92 61 100 Pa_TRINITY_DN14147_c0_g1_i7_UbxPt_XP_021004342.1_Ubx-b Pp_c95663_g1_i1_Dfd 100 100 Ca_TRINITY_DN120_c1_g4_i1_Dfd 100 Eb_TRINITY_DN1861_c7_g1_i1_Dfd 100 Cs_XP_023234845.1_Dfd-bMe_MMa11633_Dfd Ca_TRINITY_DN6462_c0_g2_i1_Ubx100 97 99 99 Is_XP_029832119.2_Dfd 91 80 Pf_TRINITY_DN11690_c0_g1_i2_Dfd 100 38 84 Eb_TRINITY_DN61_c0_g2_i4_Ubx 80 Ca_TRINITY_DN31049_c1_g1_i2_DfdEk_CCK73372.1_Dfd 100 Eb_TRINITY_DN93116_c0_g2_i3_Dfd100 Ca_TRINITY_DN120_c1_g1_i3_Ubx 68 Sm_SMAR004162_Dfd 100 Lp_XP_022243396.1_Dfd 100 76 Lp_XP_022235204.1_Dfd Me_MMa46055_Ubx 99 100 99 86 Cs_XP_023228870.1_Ubx-b 61 100 Lp_XP_013784402.2_Dfd 100 92 100 Lp_XP_013776725.2_Dfd Po_CCH51007.1_Ubx_partial 86 Lp_XP_013792158.2_Dfd 88 Ubx Dp_EFX86802.1_Ubx 92 98 97 Cs_XP_023230593.1_Dfd-a 100Me_MMa55595_Dfd Dm_NP_732173.1_Ubx 95 73 Mm_TRINITY_DN3338_c0_g3_i1_Dfd Tc_NP_001034497.1_Ubx 52 100 100Pt_XP_015913685.1_Dfd-b 100 Bm_NP_001107632.1_Ubx 75 Tu_XP_015790214.1_Dfd 100 92 Tu_XP_015790202.1_ftz-2 Tu_XP_015790297.1_ftz-1 Sm_SMAR004167_Ubx 79 61 Tu_XP_015787094.1_lab 100 Ag_TRINITY_DN82983_c3_g1_i3_Ubx 98 100 Bm_XP_037867477.1_lab Pp_c109150_g2_i4_Ubx 99 Tc_NP_001107762.1_lab 64 Dp_EFX86813.1_lab Sd_XM_035374807.1_Ubx100 100 Pan_SBLA01081258.1-c516943-516560_lab_partial 100 100 Pt_XP_015922008.1_Ubx-a 95Pa_TRINITY_DN11046_c0_g1_i1_lab 100 93Mm_TRINITY_DN2945_c0_g1_i1_lab Mm_TRINITY_DN28_c7_g1_i11_Ubx 100 94Sd_XM_035351786.1_lab Pa_TRINITY_DN5810_c0_g1_i1_Ubx 93 Pt_XP_015909180.1_lab-b 98 70 94 Me_MMa34599_Ubx Pp_c103960_g6_i1_lab Pan_SBLA01416538.1-c964848-964513_Ubx_partial96 Ag_TRINITY_DN71545_c1_g1_i1_lab 100 Me_MMa06611_abdA Ca_TRINITY_DN92496_c0_g1_i1_lab Mm_TRINITY_DN14602_c0_g1_i4_Ubx 100 99 89 Eb_TRINITY_DN21631_c1_g1_i1_lab 99 99 99 67 Pan_SBLA01092602.1-2176019-2176390_Ubx_partial Lp_XP_022244576.1_lab 100Ca_TRINITY_DN106247_c0_g1_i1_lab 44 89 100 Eb_TRINITY_DN16164_c0_g1_i2_lab Po_CCH51008.1_abdA_partial 82

Ek_CCK73377.1_abdA 68 94 Ag_TRINITY_DN80520_c5_g2_i2_lab 87 100 76 Ag_TRINITY_DN37887_c0_g1_i1_abdA_partial Pan_SBLA01123087.1-982556-982924_abdA_partialDm_NP_732176.1_abdADp_EFX86800.1_abdA 98 100

62 94 Tc_NP_001034518.1_abdA 94 80 76 Po_CCH51000.1_lab_partial 41 89 67 Lab Bm_NP_001166808.1_abdA 7 Sm_SMAR004168_abdA 9 100 98 Is_XP_029832157.1_lab 82 Pf_TRINITY_DN16093_c4_g7_i1_lab 76 69 Cs_XP_023222468.1_lab-b Ek_CCK73369.1_lab 100 100 94 Is_XP_029842575.2_abdA Sm_SMAR004159_lab 100 99 Cs_XP_023221395.1_abdA-b 100 100 98 Me_MMa55584_labMe_MMa32532_lab 95 100 Sd_XM_035350663.1_lab Lp_XP_022243120.1_abdA 73 Cs_XP_023221625.1_lab-a 100 100 Lp_XP_013776095.1_lab Lp_XP_022237373.1_abdA Me_MMa41694_abdA 80 100 Dm_NP_476613.1_lab 100 87 88 Lp_XP_013790346.1_lab 100 100 100 Cs_XP_023228869.1_abdA-a 82 Pt_NP_001310763.1_lab-a Pp_c104286_g1_i3_abdA 95 Pa_TRINITY_DN3878_c0_g1_i1_lab 100 Mm_TRINITY_DN9126_c9_g1_i1_lab Ek_CCK73374.1_ftz 88 77 97 80 Pan_SBLA01017558.1-c82241-81996_lab_partial

100 97

Sd_XM_035357390.1_abdA 55 51 Mm_TRINITY_DN24857_c0_g2_i3_abdA 90

Pt_XP_015921999.1_abdA-a 100 66

65

100 99 59 97 78 Dp_EFX86809.1_Hox3 Pa_TRINITY_DN17976_c0_g1_i3_abdAEb_TRINITY_DN61_c0_g1_i9_abdA 96

100 84 Ca_TRINITY_DN690_c0_g1_i1_abdA 85 Pf_TRINITY_DN7386_c0_g1_i1_ftz 7 Eb_TRINITY_DN864_c6_g1_i2_abdA 8 Pp_c95590_g1_i1_abdA 99 97 Ca_TRINITY_DN776_c5_g1_i1_abdA abdA 97 Ag_TRINITY_DN79526_c1_g2_i6_abdAPt_XP_015930560.1_abdA-b Eb_TRINITY_DN14061_c0_g1_i2_ftzCa_TRINITY_DN8055_c0_g1_i1_ftz

Sd_XM_035349895.1_abdA Sm_SMAR004163_ftz 100 Lp_XP_013776722.2_ftz 82

100 79

77 Mm_TRINITY_DN26258_c0_g1_i1_abdA Pp_c107945_g3_i3_Hox3 84

99

75 Pa_TRINITY_DN11599_c0_g1_i1_abdA 100

94 97 Po_CCH51005.1_ftz_partialMe_MMa55597_ftz 79

71 93 Is_XP_002406403.2_ftz 99

97 38 99 Ca_TRINITY_DN6462_c0_g1_i1_ftz 94 Ca_TRINITY_DN4729_c0_g3_i1_Hox3

E

100 100

100 Pan_SBLA01263448.1-c839378-838266_Hox3_partial T_b Ag_TRINITY_DN80542_c8_g2_i1_Hox3 Pa_TRINITY_DN9253_c0_g1_i1_Hox3 67 Mm_TRINITY_DN55812_c0_g1_i10_Hox3 Pan_SBLA01079679.1-441501-443333_pb_partial R 96 Is_XP_029832131.2_Hox3 Sd_XM_035372716.1_Hox3 1 Me_MMa24497_ftz S 100 Pan_SBLA01092602.1-1773909-1774487_abdA-1_partial I 0

N

d 0 Lp_XP_022255072.1_Hox3 99 100Pa_TRINITY_DN4746_c0_g1_i1_pb Cs_Leit_et_al_2018_ftz-a Pt_XP_015909177.1_Hox3-b 3xoH_1.17337 98 Eb_TRINITY_DN45249_c0_g2_i1_ftz X_

YTI

M 99 99 Cs_XP_023234985.1_ftz-b _ 100 100

100 bE _ Mm_TRINITY_DN9011_c0_g1_i2_pb D 99

_ aC 3xoH_1i_1g_6 Sd_XM_035375931.1_pb

_ 30 Me_MMa55591_Hox3

1N T RT Pp_c92385_g1_i1_ftz 5 77 Pp_c101164_g7_i1_Hox3

Tc_NP_001034539.1_ftz Ag_TRINITY_DN3993_c0_g1_i1_ftz_partial R 3

Bm_XP_021206502.1_zen NI 54 Pt_XP_015928816.1_pb-a 5 Ag_TRINITY_DN77199_c7_g3_i1_pb Sd_XM_035376242.1_ftz I Eb_TRINITY_DN4025_c1_g1_i1_pb 85 N

2

Pt_XP_015917916.1_ftz TI 50 100

TI 71 Cs_XP_023230602.1_pb-a Pp_c104684_g3_i1_pb Y

_Y K 1 100

D _ .

CC_kE

1 0c_4

D Me_MMa55590_pb

0

_ _

0

N

5N g c_

1

9 6 1 oH

3xoH_6 4 77 786 x 100

1 i_ 3

_ _49 100 Pf_TRINITY_DN15728_c0_g1_i1_Hox3

_1

c c

77ND_ Pa_TRINITY_DN17477_c0_g1_i1_ftz 0 100 Lp_XP_013777106.2_pb Dp_NP_477498.1_ftz Lp_XP_022244577.1_Hox3 _ Tu_XP_015787097.1_pb 53 Lp_XP_022252679.1_Hox3

g g_0 76 Tc_NP_001036813.1_zen 5

3xoH

1

3

Bm_XP_021206595.2_ftz _ _1 55a Pan_SBLA01117046.1-2197441-2199468_pb_partial 74 Tc_NP_001038090.1_zen2 i Lp_XP_013786746.1_pb i YT 100 Lp_XP_013790991.2_pb

3 1 100

_ _ 99 Pa_TRINITY_DN12900_c0_g1_i1_pb I 100

H M 100 NI 100 Mm_TRINITY_DN53186_c0_g1_i1_pb

M_e

xo RT Sd_XM_035367140.1_pb Is_XP_029832076.2_pb

3xoH 3 Pt_XP_015909179.1_pb-b _gA

M

Cs_XP_023234869.1_Hox3 Pp_c104593_g1_i1_pb Pf_TRINITY_DN15726_c3_g7_i1_pb

Dp_EFX86812.1_pb Pan_SBLA01129491.1-550743-551141_ftz_partial 79

Dm_NP_476793.1_zen

Mm_TRINITY_DN93371_c0_g1_i1_ftz_partial Cs_XP_023234832.1_pb-b 100 100 Me_MMa35910_pb Sm_SMAR015657_Hox3

Dm_NP_476794.1_zen2 99 Sm_SMAR004160_pb

Ftz Bm_XP_021206544.1_pb Tc_NP_001107807.1_mxp Ek_CCK73370.1_pb

100

Ca_TRINITY_DN34129_c2_g1_i1_pb Dm_NP_996163.1_pb

Po_CCH51002.1_Hox3_partial

Po_comp92303_c0_seq2_pb_partial bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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. Pb Hox3

Dm_NP_731111.1_bcd

2.0 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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. Wnt1Wnt2Wnt3Wnt4Wnt5Wnt6Wnt7Wnt8Wnt9Wnt10Wnt11Wnt16WntA Marpissa muscosa RTA

WGD Pardosa amentata

Pardosa pseudoannulata

Entelegynae Stegodyphus dumicola

Parasteatoda tepidariorum Araneae Synspermiata Pholcus phalangioides

Mygalomorphae Acanthoscurria geniculata Arachnopulmonata Euphrynichus bacillifer Amblypygi

Charinus acosta

Arachnida Mesobuthus martensii Scorpiones

Centruroides sculpturatus Eucherlicerata Opiliones

Cherlicerata Phalangium opilio

Parasitiformes Ixodes scapularis

Arthropoda Acariformes Tetranychus urticae Xiphosura Limulus polyphemus 6 6 4 4 5 Pycnogonida Phoxichilidium femoratum

Diptera Drosophila melanogaster

Lepidoptera Insecta Bombyx mori

Coleoptera Tribolium castaneum Mandibulata Crustacea Daphnia pulex

Myriapoda Strigamia maritima

Onychophora Euperipatoides kanangrensis Ca_TRINITY_DN14514_c0_g4_i1_Wnt6

Pf_TRINITY_DN16671_c6_g1_i15_Wnt6

Is_XP_029850753.1_Wnt6

Lp_XP_013790949.2_Wnt6_partial Wnt6

Ca_TRINITY_DN148921_c0_g1_i1_Wnt6-1

Eb_TRINITY_DN25532_c0_g1_i1_Wnt6

Mm_TRINITY_DN111554_c0_g2_i1_Wnt6

Ag_TRINITY_DN68596_c0_g1_i1_Wnt6

Pan_SBLA01575378.1_Wnt6_partial Sd_XP_035218275.1_Wnt6 Cs_XP_023219342.1_Wnt6 Po_Wnt6_clone

Pa_TRINITY_DN3753_c0_g1_i6_Wnt6 Pt_XP_015906156.1_Wnt6 Pp_c111740_g4_i1_Wnt6 Cs_XP_023228802.1_Wnt6 Wnt4 Me_MMa17732_Wnt6

Dm_NP_609108.3_Wnt6

Me_MMa40874_Wnt6

Ag_TRINITY_DN82741_c1_g1_i2_Wnt1

Tu_XP_015786865.1_Wnt6

Pf_TRINITY_DN16597_c4_g3_i1_Wnt1 Tc_NP_001107822.1_Wnt1

Bm_NP_001037315.1_Wnt1

Pan_SBLA01274480.1_Wnt4_partial

Tc_XP_015835201.1_Wnt6 100 Dm_NP_523502.1_Wnt1

100 Sm_SMAR002921_Wnt6

Pa_TRINITY_DN18193_c0_g1_i1_Wnt4

100 100

Dp_EFX86167.1_Wnt6 94 Bm_XP_037866642.1_Wnt6 100 Mm_TRINITY_DN9777_c1_g1_i4_Wnt4 Wnt1 100 100 94 Pan_SBLA01315990.1_Wnt11_partial

100 Ca_TRINITY_DN76533_c0_g1_i1_Wnt1 98 100 Sm_SMAR002920_Wnt1

98

Sd_XP_035206636.1_Wnt4 Eb_TRINITY_DN40663_c0_g1_i1_Wnt1 Pan_SBLA01282435.1_Wnt4-partial

Ek_CDI40102.1_Wnt6 Pa_TRINITY_DN19987_c0_g2_i1_Wnt4 Pa_TRINITY_DN83378_c0_g1_i1_Wnt11 Cs_XP_023228816.1_Wnt1 57 100 88 Pt_BBD75281.1_Wnt4 Tu_XP_015783539.1_Wnt4 Me_MMa40875_Wnt1 Ek_ABY60732.1_Wnt1 Mm_TRINITY_DN2819_c0_g2_i1_Wnt4 100 Dp_EFX86386.1_Wnt1 100 Ag_TRINITY_DN22838_c0_g1_i1_Wnt4

52 Mm_TRINITY_DN171_c19_g2_i4_Wnt11

Is_XP_002407192.2_Wnt1 50 Me_MMa16104_Wnt4

84 100 Ag_TRINITY_DN98907_c0_g1_i1_Wnt1 87 Is_XP_002436043.2_Wnt4 88 Sd_XP_035208146.1_Wnt4 Lp_XP_013788136.1_Wnt1_partial 100

73

Ag_TRINITY_DN66402_c1_g1_i1_Wnt4 95 92 Pt_XP_015920222.1_Wnt11-1 100 100 Eb_TRINITY_DN25384_c0_g2_i3_Wnt4Ca_TRINITY_DN9561_c0_g1_i3_Wnt4 Cs_XP_023219341.1_Wnt1 Sd_XM_035378341.1_Wnt11 76 100 100 54 Cs_XP_023222233.1_Wnt4 91 Pp_c104722_g2_i2_Wnt4 100

Ca_TRINITY_DN2244_c14_g1_i1_Wnt1Po_Wnt1_clone 76 100 100 Me_MMa13725_Wnt1 Ca_TRINITY_DN7758_c0_g1_i1_Wnt4 100 Eb_TRINITY_DN32332_c8_g1_i1_Wnt1 Eb_TRINITY_DN25384_c0_g1_i1_Wnt4 100 100

100 100 Pan_SBLA01551669.1_Wnt1_partialMm_TRINITY_DN19470_c0_g1_i5_Wnt1 100 Pf_TRINITY_DN14285_c0_g1_i1_Wnt4 Pt_XP_015906154.1_Wnt1Pp_c104855_g3_i1_Wnt1 Lp_XP_022249841.1_Wnt4 Sd_XP_035218273.1_Wnt1 72 99 Ag_TRINITY_DN69200_c0_g2_i1_Wnt11 Pan_SBLA01068646.1_Wnt1_partial Pp_c545_g1_i1_Wnt11 100 95 70 71 58 100 98 Pan_SBLA01236476.1_Wnt11_partial T_aPR 91 60 Sm_SMAR013747_Wnt4 TINI 87 Y 100 84 70 100 98 56 Po_Wnt4_clone Pa_TRINITY_DN4436_c0_g1_i1_Wnt11 4986ND_ 68 100 c_ 87 85 Sd_XM_035371679.1_Wnt11 _mD _2 100 100 95 N 1g 100 82 Ca_TRINITY_DN124475_c0_g1_i1_Wnt1198 4_P i_ 87 100 Mm_TRINITY_DN2809_c0_g1_i5_Wnt11 967 74 99 Dp_EFX72339.1_Wnt4 24 Ek_CDI40100.1_Wnt4 .1 1tnW_1 100

W_ 92 n 96 Wnt11 5t Pt_XP_015916686.1_Wnt11

99 98 96 98 Pf 100 Pp_c110869_g1_i1_Wnt11 B _cTX X_mP 79_P4 100

0_3 6 1 N D _ Y TINIR T_ Me_MMa52170_Wnt11 87 0 7 _1.48W 103 80 17 c_3 3.1 5tn _0 100 _W Cs_XP_023228131.1_Wnt11 nt 100 Eb_TRINITY_DN25108_c0_g1_i1_Wnt11 5 Ca_TRINITY_DN84356_c0_g1_i1_Wnt11 Pf_TRINITY_DN16417_c3_g7_i1_Wnt11 nW_1i_1g 98 100 5t 4IDC_kE0 Ek_CDI40106.1_Wnt11 Lp_XP_022245914.1_Wnt5

W_1.101 Lp_XP_022246278.1_Wnt5_partialDp_EFX66479.1_Wnt569 5tn 98 Lp_XP_013792286.2_Wnt5_partial 70 Sm_SMAR008487_Wnt5 80 Lp_XP_022235854.1_Wnt11 Tu_XP_015794709.1_Wnt5 36 97 Lp_XP_022248877.1_Wnt11 98

64 Lp_XP_022250690.1_Wnt11 6 Lp_XP_022254081.1_Wnt11 7 57 100 100 65 100 100 1 00 80 Is_XP_002434188.1_Wnt11 Po_Wnt5_clone 100 100 Lp_XP_022240862.1_Wnt5 63 94 Po_Wnt11_202686_degenerate_rc_PRT Wnt5 Lp_XP_013792736.1_Wnt5 68 98 Me_MMa16972_Wnt11 99 Dp_EFX77586.1_Wnt11 Cs_XP_023240364.1_Wnt11 Bm_XP_004924665.2_Wnt11 Is_XP_029847985.1_Wnt5Lp_XP_013783628.2_Wnt5 100 100 99 93 71 Cs_XP_023215183.1_Wnt5 Tc_XP_015835988.1_Wnt11 100 72 99 Me_MMa45995_Wnt5 99 96 Ca_TRINITY_DN27029_c0_g1_i1_Wnt5100 100 Eb_TRINITY_DN1299_c0_g1_i1_Wnt5 100 98 Pa_TRINITY_DN5325_c0_g1_i4_Wnt7 90 100 Ag_TRINITY_DN64024_c0_g1_i1_Wnt5 Pan_SBLA01298555.1_Wnt7_partial 100 100 Pp_c100246_g1_i2_Wnt5 Mm_TRINITY_DN33882_c0_g1_i1_Wnt7 100 100 Sd_XP_035223051.1_Wnt5 100 Pt_NP_001310746.1_Wnt7-2 100 100 88 Sd_XM_035361740.1_Wnt7 Pt_NP_001310745.1_Wnt5 100 61 Pp_c103422_g2_i1_Wnt7 Mm_TRINITY_DN18793_c0_g1_i1_Wnt5 63 100 62 Ag_TRINITY_DN70434_c1_g2_i1_Wnt7 Pan_SBLA01507739.1_Wnt5_partial 100 Po_Wnt7_full_ORF 100 Eb_TRINITY_DN2459_c0_g2_i2_Wnt7 Pa_TRINITY_DN1669_c0_g1_i1_Wnt5 Ca_TRINITY_DN14295_c1_g2_i2_Wnt7 100 100 Tc_KYB26594.1_WntA Pa_TRINITY_DN18962_c0_g1_i5_Wnt7 93 100

Bm_XP_021209300.1_WntA 93 100 Pan_SBLA01420016.1_Wnt7_partial

96 100 Mm_TRINITY_DN18996_c1_g3_i3_Wnt7 Dp_EFX69968.1_WntA 96

97 50 99 100 Pt_NP_001310739.1_Wnt7-1 Pf_TRINITY_DN11305_c0_g1_i3_WntA 96 Eb_TRINITY_DN2459_c0_g1_i1_Wnt7 Sm_SMAR015773_WntA 100 Ag_TRINITY_DN75361_c0_g1_i1_Wnt7Pp_c47853_g1_i2_Wnt7 77 60 99 Ca_TRINITY_DN7933_c0_g1_i2_Wnt7 100 76 92 Sd_XP_035210859.1_Wnt7 Ek_CDI40108.1_WntA 72 Me_MMa04980_Wnt7 100 99 100 82 100 53 Is_XP_002402520.2_WntA 83 100 100 33 100 Cs_XP_023220709.1_Wnt7 100 78 100 Me_MMa17290_Wnt7

Cs_XP_023215187.1_Wnt7

100 83 98 100 0 Lp_XP_013773810.1_Wnt7 100 80 100 01 100 100 39 81 Tu_XP_015794728.1_WntALp_XP_022253065.1_WntA L 100 L _pX 99 56 _p P_ Lp_XP_022247569.1_WntA XP 02 kE 0_ 22 31 51 7 70 Lp_XP_022246203.1_WntA_partial 100 100 100 1.0 67 001 _ Lp_XP_022238400.1_WntA 0104IDC_ 3 W_1.73339nWt Po_WntA_zus n 7_ 97 87 t pa _7p itr 100 7tnW_1.001 a tr al 90 Sm_SMAR012534_Wnt7 lai 100 100 93 Lp_XP_013783194.2_WntA 100 Tc_XP_008196351.1_Wnt7 pL Wnt7 85 Dp_EFX66449.1_Wnt7 X_P Me_MMa47532_WntA 0_1 Cs_XP_023218523.1_WntA Ek_CDI40099.1_Wnt2 73

100 85 87 310_PX_pL78 66 100 Lp_XP_022257593.1_Wnt2Me_MMa37570_Wnt2 100 .7101 Cs_XP_023224164.1_Wnt2 025874 _ 100 100 nW 81 Pa_TRINITY_DN7884_c0_g1_i1_Wnt16 7t 100 99 100 100100 Pp_c100839_g1_i1_Wnt2 Ca_TRINITY_DN12906_c4_g1_i1_WntAPp_c105403_g1_i3_WntA Mm_TRINITY_DN35858_c0_g1_i7_Wnt16 95 95 100 Sm_SMAR004376_Wnt2 100 laitra p _ 7tn W _ 1. WntA Ag_TRINITY_DN70434_c1_g1_i1_Wnt2 Dp_EFX87200.1_Wnt2 100 Eb_TRINITY_DN7856_c0_g1_i1_WntA Pt_NP_001310769.1_Wnt16Pan_SBLA01200618.1_Wnt16_partialDm_NP_476810.1_Wnt7 100 Ag_TRINITY_DN60488_c0_g1_i1_Wnt16 laitra p _ 7tn W _ 1.7 0 0 9 8 7 3 1 0 _ P X _ p L Pt_NP_001310740.1_Wnt2 Pp_c111004_g2_i1_Wnt16 100Me_MMa00727_Wnt16 Pf_TRINITY_DN16576_c3_g2_i1_Wnt7 Cs_XP_023224171.1_Wnt16 99 100 Ag_TRINITY_DN70458_c0_g1_i1_WntA Eb_TRINITY_DN4235_c0_g1_i1_Wnt16 82 100 Bm_XP_021208534.1_Wnt7 Ca_TRINITY_DN22656_c0_g1_i2_Wnt16 Sd_XP_035205952.1_WntA Dp_EFX82994.1_Wnt16 89 Ek_CDI40107.1_Wnt16 Pa_TRINITY_DN22647_c0_g1_i5_Wnt2 Pt_XP_015915580.1_WntA 86 Is_EEC17948.1_Wnt7 Lp_XP_022249784.1_Wnt2 85 100

Eb_TRINITY_DN13499_c1_g1_i23_Wnt2 Pf_TRINITY_DN14850_c0_g1_i2_Wnt16 Pf_TRINITY_DN15706_c6_g1_i1_Wnt2

100 Lp_XP_022242527.1_Wnt16Lp_XP_013792115.1_Wnt16_partial Mm_TRINITY_DN24839_c0_g1_i2_Wnt2 Mm_TRINITY_DN193136_c0_g1_i1_WntA 100 Sd_XP_035225808.1_Wnt2 Ca_TRINITY_DN6257_c1_g1_i1_Wnt2 Po_Wnt16_clone Sd_XP_035210368.1_Wnt16 100 Lp_XP_022246054.1_Wnt16 Pan_SBLA01324069.1_Wnt2_partial Pan_SBLA01407515.1_WntA_partial 100

Pa_TRINITY_DN6044_c0_g1_i2_WntA 97

97 Pan_SBLA01407515.1_WntA_partial 100

100 96 Wnt2 100

86 Tu_XP_025017632.1_Wnt16

83

100

100

Ek_CDI40105.1_Wnt10 99

Dp_EFX86388.1_Wnt10

97 97 Is_XP_029828193.1_Wnt16

82 80

87

68 82 Wnt16 Po_Wnt10_clone Dm_NP_609109.3_Wnt10 94

Lp_XP_022252175.1_Wnt16

75

Ca_TRINITY_DN2407_c0_g1_i1_Wnt10 100

Tc_XP_008193179.1_Wnt10 100

Eb_TRINITY_DN12659_c0_g1_i4_Wnt10 100

Sm_SMAR002922_Wnt10 100

100

97

Lp_XP_013783076.2_Wnt8

Ag_TRINITY_DN77370_c0_g1_i1_Wnt8

100

Dp_EFX83364.1_Wnt8 Eb_TRINITY_DN15273_c0_g1_i1_Wnt8

Bm_XP_037866617.1_Wnt10 Sd_XP_035224391.1_Wnt8 Pa_TRINITY_DN49042_c0_g1_i3_Wnt8 100

100

Is_EEC10449.1_Wnt9 Ca_TRINITY_DN33459_c0_g1_i2_Wnt8 Ek_CDI40104.1_Wnt9

Mm_TRINITY_DN8243_c0_g1_i2_Wnt8

Dm_NP_476972.2_Wnt9

Tc_XP_971439.1_Wnt8 Pan_SBLA01132800.1_Wnt8_partial

Ca_TRINITY_DN65449_c0_g1_i1_Wnt9 Eb_TRINITY_DN135499_c0_g1_i1_Wnt9

Pp_c93728_g1_i2_Wnt8 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

Dp_EFX86385.1_Wnt9

Pt_NP_001310760.1_Wnt8

Pf_TRINITY_DN12881_c0_g1_i2_Wnt8

Me_MMa40873_Wnt9 Po_Wnt8_clone Wnt10 Cs_XP_023228817.1_Wnt9

Tc_XP_015835609.1_Wnt9

Po_Wnt9_clone

Bm_XP_012548374.2_Wnt9

Pf_TRINITY_DN16417_c3_g13_i2_Wnt9_partial

Is_Janssen_et_al_2010_Wnt8

Sm_SMAR014558_Wnt9 Tu_XP_015790171.1_Wnt8 Wnt8 Wnt9

1.0 Dm_NP_650272.1_Wnt8 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. 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.

FzI FzII FzIIIFzIV Marpissa muscosa RTA

WGD Pardosa amentata

Pardosa pseudoannulata

Entelegynae Stegodyphus dumicola

Parasteatoda tepidariorum Araneae Synspermiata Pholcus phalangioides

Mygalomorphae Acanthoscurria geniculata Arachnopulmonata Euphrynichus bacillifer Amblypygi

Charinus acosta

Arachnida Mesobuthus martensii Scorpiones Centruroides sculpturatus Eucherlicerata Opiliones

Cherlicerata Phalangium opilio

Parasitiformes Ixodes scapularis

Arthropoda Acariformes Tetranychus urticae Xiphosura Limulus polyphemus 5 5 Pycnogonida Phoxichilidium femoratum

Diptera Drosophila melanogaster

Lepidoptera Insecta Bombyx mori

Coleoptera Tribolium castaneum Mandibulata Crustacea Daphnia pulex

Myriapoda Strigamia maritima

Onychophora Euperipatoides kanangrensis Tu_XP_015781163.1_Fz2 Tu_XP_015790096.1_Fz2

Lp_XP_022247003.1_Fz2 Mm_TRINITY_DN167403_c0_g1_i1_Fz2

Lp_XP_022253757.1_Fz2

Fz2 Lp_XP_022235618.1_Fz2 100

Lp_XP_022255782.1_Fz2

Po_comp186257_c1_seq1_Fz2

Lp_XP_022243301.1_Fz2

Eb_TRINITY_DN119144_c0_g1_i1_Fz2

Dp_EFX87638.1_fz2 Ca_TRINITY_DN28687_c0_g1_i1_Fz2 Bm_XP_021204477.1_Fz2

Cs_XP_023229226.1_Fz2 Ca_TRINITY_DN40043_c0_g1_i1_Fz2

Dm_NP_001262037.1_Fz2

Is_XP_029824354.1_Fz2

100 Mm_TRINITY_DN42824_c2_g1_i1_Fz2 100 Sd_XP_035216180.1_Fz2 Tc_EFA01325.1_Fz2

100 99

95

100 93 Ek_CRI73784.1_Fz1

Pa_TRINITY_DN36274_c0_g1_i1_Fz2Pan_SBLA01494123.1_Fz2 Sm_SMAR014833_Fz1Pp_c106393_g7_i1_Fz1Pt_XP_015922960.1_Fz1

95 93

100 82 Pa_TRINITY_DN8551_c0_g1_i1_Fz1

95 99 Pan_SBLA01061736.1_Fz1 96 91 79 99 98 100 100 Mm_TRINITY_DN207991_c0_g1_i1_Fz1 Sd_XP_035207990.1_Fz1_partial 93 99 Pt_XP_015922948.1_Fz2 86 99 Sd_XP_035219450.1_Fz1 Ag_TRINITY_DN79654_c0_g1_i1_Fz2 100 100 100 99 Fz1 83 79 Ag_TRINITY_DN36474_c0_g1_i1_Fz1

99 100 Ca_TRINITY_DN15108_c0_g1_i1_Fz1 Pf_TRINITY_DN16650_c1_g1_i1_Fz2Pp_c93104_g3_i1_Fz2 100 Eb_TRINITY_DN2020_c0_g1_i2_Fz1 54

82 Cs_XP_023229313.1_Fz1 93 Sm_SMAR012389_Fz2 100 100Me_AYEL01056881.1_Fz1Me_AYEL01067974.1_Fz1_partial 100

Tu_XP_025016759.1_Fz4 100 77 91 Ca_TRINITY_DN6276_c6_g1_i1_Fz1 Ek_CRI73785.1_Fz2 100 75 Eb_TRINITY_DN142670_c0_g1_i1_Fz1

Pf_TRINITY_DN1931_c0_g1_i1_Fz4 Po_comp184244_c0_seq2_Fz1 100 99 Is_XP_029849982.1_Fz1 97 100 4922 F_1.z1 Lp X__P013791589.1_Fz4 Lp_XP_01378 97 Lp_XP_013792939.1_Fz4 100 100 94 100Lp_XP_022252858.1_Fz1 100 88 z4 Lp X_P 0_1 737985 F_1.7 L 100 p_X _P0137863351.F_z1 91 100 Po_comp187854_c1_seq2_Fz4 Lp_XP_013788049.1_Fz1 100 76 Lp_XP_013788951.1_Fz1 Is_XP_002402968.1_Fz4 100 89 95 Me_AYEL01071131.1_Fz4 Dp_EFX86267.1_Fz1 82 100 99 Cs_XP_023220057.1_Fz4 Bm_XP_004929280.1_Fz1 100 100 100 98 Ek_CRI73786.1_Fz3 72 99 84 Me_AYEL01075372.1_Fz4 Pf_TRINITY_DN15944_c8_g3_i1_Fz1Tc_EFA04653.1_Fz1Dm_NP_001261836.1_Fz1 98 100 75 Cs_XP_023233147.1_Fz4 100 99 76 70 Sm_SMAR007293_Fz3 100 100 72 Eb_TRINITY_DN127241_c0_g1_i1_Fz4 100 100 98 Ca_TRINITY_DN158988_c0_g1_i1_Fz3 Ag_TRINITY_DN72984_c0_g1_i1_Fz3Pp_c111597_g3_i1_Fz3 66 Eb_TRINITY_DN7482_c0_g1_i1_Fz3 Cs_XP_023221477.1_Fz3 Ca_TRINITY_DN127345_c0_g1_i1_Fz4 79 100 Me_AYEL01075007.1_Fz3 93 100 100 Ek_CRI73787.1_Fz4 Ca_TRINITY_DN159346_c0_g1_i1_Fz3 Sd_XP_035206735.1_Fz4 100 100 Eb_TRINITY_DN1947_c0_g1_i1_Fz3

100 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.177725; this version posted April 26, 2021. The copyright holder for this preprint (which 100 100 was not certified by peer review) is the author/funder. All rightsPt_XP_015910102.1_Fz4-2 reserved. No reuse allowed without permission. 100 Ag_TRINITY_DN82058_c5_g1_i1_Fz4 Lp_XP_022240618.1_Fz3 100 Is_XP_002400653.2_Fz3 Eb_TRINITY_DN17745_c0_g1_i1_Fz4 98

Lp_XP_013781532.1_Fz3Lp_XP_013778946.2_Fz3

Ca_TRINITY_DN24116_c0_g2_i1_Fz4

Sm_SMAR009650_Fz4

Pf_TRINITY_DN10278_c0_g1_i2_Fz3 Pp_c102918_g1_i1_Fz4 97 Pan_SBLA01585531.1_Fz4

Mm_TRINITY_DN150_c35_g1_i1_Fz4 Fz4 Sd_XP_035227549.1_Fz4

Pa_TRINITY_DN1229_c0_g1_i1_Fz4 Ag_TRINITY_DN81345_c1_g1_i1_Fz4

Pt_XP_015911110.1_Fz4-1

Tc_EFA09255.1_Fz4 Fz3

Bm_XP_012545937.1_Fz4

Dm_NP_511068.2_Fz4

Bm_XP_021202154.2_Fz3

Dm_NP_001096859.1_Fz3

0.8