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Neglected no longer: Phylogenomic resolution of higher-
level relationships in Solifugae
1Siddharth S. Kulkarni, 1Hugh G. Steiner, 2Erika L. Garcia, 3Hernán Iuri, 2R. Ryan
Jones, 4Jesús A. Ballesteros, 1Guilherme Gainett, 5Matthew R. Graham, 6Danilo Harms,
7Robin Lyle, 3Andrés A. Ojanguren-Affilastro, 8Carlos E. Santibañez-López, 9Gustavo
Silva de Miranda, 2Paula E. Cushing, 10Efrat Gavish-Regev, 1Prashant P. Sharma
1 Department of Integrative Biology, University of Wisconsin–Madison, Madison, WI
53706, USA
2 Department of Zoology, Denver Museum of Nature & Science, Denver, CO 80205,
USA
3 División de Aracnología, Museo Argentino de Ciencias Naturales “Bernardino
Rivadavia”, Buenos Aires 1405DJR, Argentina
4 Department of Biology, Kean University, Union, NJ 07083, USA
5 Department of Biology, Eastern Connecticut State University, Willimantic, CT 06226,
USA
6 Museum of Nature Hamburg - Zoology, Department of Invertebrates, Leibniz Institute
for the Analysis of Biodiversity Change, Hamburg, Germany
7 Biosystematics: Arachnology, ARC—Plant Health and Protection, Pretoria, South
Africa
8 Department of Biology, Western Connecticut State University, Danbury, CT 06810,
USA
9 Department of Entomology, National Museum of Natural History, Smithsonian
Institution, Washington, DC, 20560, USA
10 The National Natural History Collections, The Hebrew University of Jerusalem, 91904
Jerusalem, Israel
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Abstract
Considerable progress has been achieved in resolving higher-level relationships of
Arthropoda in the past two decades, largely precipitated by advances in sequencing
technology. Yet, dark branches persist in the arthropod tree of life, principally among
groups that are difficult to collect, occur in cryptic habitats, or are characterized by
minute body size. Among chelicerates, the mesodiverse order Solifugae (commonly
called camel spiders or sun spiders) is one of the last orders of Arachnida that lacks a
higher-level phylogeny altogether and has long been characterized as one of the
“neglected cousins”, a lineage of arachnid orders that are comparatively poorly studied
with respect to evolutionary relationships. Though renowned for their aggression,
remarkable running speed, and adaptation to arid habitats, inferring solifuge
relationships has been hindered by inaccessibility of diagnostic characters in most
ontogenetic stages for morphological datasets, whereas molecular investigations to
date have been limited to one of the 12 recognized families. In this study we generated
a phylogenomic dataset via capture of ultraconserved elements (UCEs) and sampled all
extant families. We recovered a well-resolved phylogeny of solifuge families, with two
distinct groups of New World taxa nested within a broader Paleotropical radiation. To
provide a temporal context to solifuge diversification, we estimated molecular
divergence times using fossil calibrations within a least-squares framework. Solifugae
were inferred to have radiated by the Permian, with divergences of most families dating
to the post Paleogene-Cretaceous extinction. These results accord with a diversification
history largely driven by vicariance as a result of continental breakup.
Keywords: Ultraconserved | arachnid | Pangea | diversification | molecular dating
Introduction
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Chelicerata is an ancient, monophyletic group of arthropods that is characterized by
extensive diversity, high body plan disparity among their different orders, and inclusion
of numerous charismatic taxa, such as spiders, scorpions, and horseshoe crabs.
Despite their considerable diversity, nearly every group of chelicerate orders has
benefitted from recent advances in molecular phylogenetics, genomics, and renewed
interest in evolutionary dynamics. The past decade alone has witnessed the first
molecular phylogenetic hypotheses for several orders, such as Scorpiones, Ricinulei
(hooded tick-spiders), Palpigradi (microwhip scorpions), Thelyphonida (vinegaroons),
Schizomida (short-tailed whip scorpions), and Amblypygi (whip spiders) (Murienne et al.
2013a; Giribet et al. 2014; Fernández and Giribet 2015; Sharma et al. 2015; Clouse et
al. 2017). More diverse chelicerate groups, such as Acari (mites), Araneae (spiders),
Opiliones (harvestmen), and Pseudoscorpiones, have witnessed a surge of evolutionary
inquiry in the past ten years (Hedin et al. 2012; Bond et al. 2014; Fernández et al. 2014;
Garrison et al. 2016; Fernández et al. 2017; Klimov et al. 2017; Santibáñez-López et al.
2019; Benavides et al. 2019a; Kulkarni et al. 2020; Ballesteros et al. 2021; Kallal et al.
2021; Santibáñez-López et al. 2022). This decade is also notable for the completion of
the first genomes for several chelicerate orders (Sanggaard et al. 2014; Gulia-Nuss et
al. 2016; Hoy et al. 2016; Kenny et al. 2016; Schwager et al. 2017; Gainett et al. 2021;
Ontano et al. 2021). These advances have revolutionized modern views of chelicerate
phylogeny and evolution, prompting reevaluations of historical paradigms of habitat
transition and evolution of complex characters (Bond et al. 2014; Fernández et al. 2014;
Sharma et al. 2014; Ballesteros and Sharma 2019; Ontano et al. 2021; Ballesteros et al.
2022).
Solifugae, variously known as “camel spiders” or “sun spiders”, is a mesodiverse group
(in comparison with other arachnid groups) that currently includes ca. 1,200 species
classified in 12 families and 144 genera (World Solifugae Catalog 2022). Notorious for
their fearsome appearance, large chelicerae (relative to body size), and high bite force,
(Meijden et al. 2012), solifuges are one of seven chelicerate orders that are commonly
referred to as “the neglected cousins” in the arachnological community, due to a dearth
of systematic and phylogenetic studies (Harvey 2002). Various characteristics of
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solifuges distinguish them from other arachnids, such as a robust pair of two-segmented
chelicerae, adhesive structures on the termini of the pedipalps for prey capture, the
presence of malleoli (“racquet organs”, used for chemoreception and analogous to the
pectines of scorpions) on the ventral surface of leg IV (Brownell & Farley 1974; Cushing
et al. 2005; Willemart et al. 2011), and an extensive tracheal system with additional
stigmata (Franz-Guess & Starck 2016). The last of these is understood to facilitate the
rapid running speed of solifuges, as well as their inhabitation of some of the driest
habitats on Earth. Historically, solifuges were thought to be closely related to
pseudoscorpions (Weygoldt and Paulus 1979; Shultz 2007), a hypothesis that has since
been contradicted by sperm ultrastructure (Alberti and Peretti 2002), rare genomic
changes and the ensuing placement of pseudoscorpions as the sister group of
scorpions (Ontano et al. 2021); but see (Michalski et al. 2022). Solifugae has more
recently been recovered as part of a clade with acariform mites (Poecilophysidea), and
possibly also palpigrades (Cephalosomata) (Pepato et al. 2010; Ballesteros et al. 2022).
A close relationship of these three orders is supported by the arrangement of the
anterior body segments and the structure of the coxal glands, and is recovered by a
subset of molecular analyses that have emphasized dense taxonomic sampling (Pepato
et al. 2010; Dunlop et al. 2012; Ballesteros et al. 2019, 2022).
By contrast to their placement in higher-level chelicerate phylogeny, the internal
relationships of Solifugae remain virtually unknown (Harvey 2002). A classification of
the twelve extant families was proposed by Roewer (1934), based on overall similarity
of characters that are, in turn, highly variable across genera and species; this
classification largely remains in place (World Solifugae Catalog 2022). Beyond the
incidence of poorly delimited higher-level taxa, a peculiarity of solifuge systematics is
the concentration of diagnostic characters in the adult males of many lineages—in some
groups, juveniles cannot be reliably assigned even to a specific family. As a result, few
attempts have been made to infer phylogenetic relationships using either morphological
or molecular datasets. Rhagodidae was suggested to be well-separated from the
remaining solifuge families on the basis of its peculiar morphology, but this inference
was not based on formal analyses and the polarity of these morphological characters is
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unknown (Roewer 1934). Investigations of specific character systems have documented
a large diversity, such as the flagellum of the male chelicera and the histology of sperm
ultrastructure, but these data have not been applied toward the goal of formal inference
of evolutionary relationships (Klann et al. 2009, 2011; Bird et al. 2015). Although
molecular data offer considerable advantages over anatomical datasets for phylogenetic
study of enigmatic groups like Solifugae, few works have addressed internal
relationships within this order. For example, RADseq data supported the monophyly of
genus A recent analysis of Eremocosta and suggested post glacial colonizations in
North American deserts (Santibáñez-López et al. 2021). A recent analysis of A recent
analysis of Iranian solifuges based on one mitochondrial gene was able to sample
seven families and the ensuing topology suggested the paraphyly of at least three
families, but without significant nodal support for most interfamilial relationships
(Maddahi et al. 2017). The only multilocus dataset applied to solifuge relationships
examined the phylogeny of the North American family Eremobatidae and this four
Sanger locus-based analysis uncovered extensive non-monophyly of constituent
genera, together with limited nodal support for deep nodes (Cushing et al. 2015).
Given the presumably ancient diversification of Solifugae, as inferred from the
appearance of crown group lineages by the Mesozoic and multiple fossil genera
(Dunlop 2010, World Solifugae Catalog 2022), bridging the knowledge gaps in solifuge
higher-level phylogeny requires the application of phylogenomic datasets and tissue
sampling from rare, often aging, tissue collections. We therefore amassed a set of 120
solifuge species drawn from natural history collections around the world and sequenced
these for a chelicerate-specific suite of ultraconserved elements (UCEs), an approach
notable for its demonstrable capability to accommodate even highly degraded tissues.
Here, we provide a robustly resolved phylogenomic tree of Solifugae, in tandem with
molecular dating efforts to provide a temporal context to solifuge diversification.
Results
We compiled a comprehensive data set with high-quality ultraconserved profiles
spanning all extant chelicerate orders, represented by 129 taxa. In this data set,
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Solifugae was represented by 107 taxa, amounting to almost 10% of the global fauna
(World Solifugae Catalog 2022), of which we generated ultraconserved element (UCE)
libraries for 90 (84%) and in silico extracted UCEs from existing UCE libraries,
transcriptomes or genomes for the remainder. Selection of outgroups prioritized the
sampling of basal splits, to facilitate node calibration in molecular dating. For each of
these thresholds for probe-to-library identity, we analyzed a family of matrices
assembled by varying taxon occupancy thresholds, from 10% to 50% occupancy. In
addition, to mitigate the impact of sparsely represented terminals, we created two
additional families of matrices using only terminals with >100 UCE loci and varying
taxon occupancy thresholds, both for the liberal and stringent thresholds for probe-to-
library identity.
Phylogenomic analyses
Maximum likelihood analyses based on 521 loci (25% occupancy; 65% probe-to-library
identity threshold) recovered a tree topology that divided solifuge families into two major
clades (which we refer as Clade I and II in the following text, see Figure 5). All families
except Ammotrechidae and Daesiidae were recovered as monophyletic with support,
barring Melanoblossidae, represented herein by a single terminal (Figure 2). To rule out
the possibility of systematic bias driven by compositional heterogeneity, GC-base
proportions were mapped on the 25% occupancy phylogeny. Only the outgroup
terminal, the palpigrade Eukoenenia spelaea exhibited a high proportion of GC-content;
however, excluding this taxon from the analysis did not affect the interfamilial
relationships of Solifugae.
The backbone tree topology of Solifugae was well-resolved and the basal split between
the two clades was invariably recovered across analyses (Figures 2, 3). Mummuciidae
was almost always nested within Ammotrechidae, rendering the family paraphyletic;
only with a matrix filtered for taxon occupancy over 40% was Ammotrechidae recovered
as monophyletic, albeit without support (Figures 2, 3). Melanoblossiidae was nested
within the Daesiidae I clade with 25% and 35% occupancies (both poorly supported)
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and with 40% occupancy, the same relationship received high support (99% ultrafast
bootstrap) whereas with 10% occupancy, it was placed as a sister group of a clade that
included Daesiidae, Mummuciidae and Ammotrechidae (poorly supported).
Across the >100 locus data sets, Mummuciidae was nested within Ammotrechidae in
phylogenies inferred from 10-25% occupancy matrices. The remaining, more complete,
but compact data sets (35-45%), recovered Mummuciidae as a sister group of
Ammotrechidae with the exception of Ammotrechidae sp. SOL007. Only in the 50%
occupancy data set was Ammotrechidae recovered as monophyletic (Figures S4-S9).
Influence of stringent homology filtering
Matrices based on stringent filtering (80% identity and coverage probe-to-library match)
discarded many loci, resulting in a more compact and denser data set across different
occupancies. At occupancies above 20%, some terminals were dropped because the
stringency of this filter did not recover any UCE locus for those taxa. Nevertheless, at
least one taxon representing each currently recognized family was present in each
occupancy data set, thereby not compromising the higher-level taxon sampling. The
tree topologies recovered for matrices constructed under this threshold recovered
similar relationships as previously reported. Gylippidae was recovered as paraphyletic
in 10-25% occupancy matrices, but this is likely attributable to low locus representation
(12 UCE loci for Gylippus fernangensis) at these occupancies. Among Solifugae, most
interfamilial relationships were similar to that of the 25% occupancy phylogeny (Figure
3, S24-28). In the >100 locus data sets compiled using 80% homology mapping filter,
most interfamilial relationships were similar to the 25% occupancy phylogeny. However,
in ≥35% occupancy data sets, some outgroup taxa represented by very few loci were
placed inside Solifugae. If these erroneous branches were to be pruned, solifuge
interfamilial relationships continued to be robust even at 50% occupancy, which
corresponds to a matrix of just 76 UCE loci.
Divergence dating and biogeographic analyses
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Divergence date optimizations calibrated with fossil age estimates in a least-squares
(LSD2) framework, with and without ingroup fossils, recovered some non-overlapping
age ranges. Diversification of the crown group Solifugae was estimated within the
Carboniferous (344-305 Ma). Clade I diversified during the Permian-middle Triassic
period (284-232 Ma), whereas Clade II diversified during the late Permian-late Triassic
period (268-205 Ma). The MRCA (most recent common ancestor) of all families in Clade
I, along with Ceromidae, Daesiidae III and Mummuciidae, originated in the Paleogene or
earlier. The MRCAs of Melanoblossidae, Daesiidae I, Daesiidae II, Ammotrechidae I,
Ammotrechidae II and Ammotrechidae III had a Cretaceous origin (Figure 4). Exclusion
of the two Solifugae fossils recovered a more recent age range for all nodes, including a
wider range of Carboniferous to Permian (334-250 Ma) age for the MRCA of Solifugae
(Figure 4).
To infer ancestral areas and reconstruct the biogeographic history of Solifugae, we
analyzed the dated tree topology in tandem with five broad geographic areas using the
RASP software (Figure 5). Given recent debates about the statistical comparability of
models with and without the jump (j) parameter (Ree and Sanmartín 2018, Matze 2022),
we trialed best-fitting models both with and without j. Historical biogeographic area
optimizations were assessed by the best fitting model using RASP on our 25%
occupancy data set. The model with highest AICc weight was the DEC+j (0.51), closely
followed by DIVA-like+j (0.48). Among models without the jump parameter, the DEC
model was closely followed by the DIVA-like model. Both DEC and DEC+j models
recovered a combination of Turanian, African tropics, Neotropical and Mediterranean
regions as the area for the most recent common ancestor (MRCA) of Solifugae (Figure
5). The ancestral area for Clade I was a combination of Turanian, Neotropical and
Mediterranean (i.e., fragments of Laurasia), whereas for Clade II, the Afrotropics (i.e., a
constituent of Gondwana) was recovered using both models (Figure 5). Most family
distributions were limited to a single biogeographic region. One group of the polyphyletic
Daesiidae (Daesiidae III) is distributed within Central Chile and the Patagonian (CCP)
region and the other two groups have an African origin (Figure 5). At the crown node of
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the CCPian and Neotropical Ammotrechidae III clade, the DEC+j model optimized CCP
as the ancestral area, which implied a dispersal event from CCP to the Neotropics. The
exclusion of the jump parameter optimized a combination of CCP and Neotropical
region as the ancestral area, thus implying a vicariance event dividing the descendant
lineages (Figure 5). Seven dispersal events were implied by the DEC+j model (marked
with red lines in Figure 5). These dispersals included three events between the
Mediterranean region and African tropics; two between Neotropical and the CCP
regions during the Paleogene period; one dispersal event from CCP to the Nearctic
region during the Paleogene-Cretaceous boundary; and one African tropics to CCP
regions during the Jurassic period were implied by the DEC+j model. However, the DEC
model implied a vicariance event at these nodes. Overall, the DEC+j model favored a
single area as origin where the descendent lineages have different regions implying a
dispersal event. The exclusion of the jump parameter optimized a combination of two or
more regions at the same ancestral nodes. Alternative coding of areas by continents for
extant taxa recovered Asia and Africa as the ancestral areas for Clade I and Clade II,
respectively. A further broader area coding by supercontinent recovered Laurasia as the
ancestral area for Clade I and Gondwana for Clade II (Figures S30-31).
Discussion
In a recent exploration of long branch attraction effects in chelicerate phylogeny,
(Ontano et al. 2021) showed that taxonomic undersampling, and specifically, omitting
the representation of basal nodes, exacerbated topological instability of fast-evolving
arachnid orders like pseudoscorpions. Using a rare genomic change as a benchmark
for phylogenetic accuracy, these analyses demonstrated that taxonomic sampling
(especially of slowly-evolving and basally branching groups) outperformed other
approaches to mitigating long branch attraction, such as increasing matrix
completeness, using coalescent-aware species tree approaches, filtering by
evolutionary rate, and implementing site heterogeneous models in concatenated
matrices. Ontano et al. (2021) reasoned that applying this remedy to other fast-evolving
orders may greatly stabilize chelicerate phylogeny, given that at least four orders exhibit
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a propensity for long branch attraction artifacts in Chelicerata (Ballesteros and Sharma
2019; Ballesteros et al. 2019; Ontano et al. 2022). This proposed remedy for long
branch attraction is greatly potentiated by the availability of higher-level phylogenies for
unstable groups, such as Acariformes (Klimov et al. 2017) and Palpigradi (Giribet et al.
2014). However, representation of solifuges in phylogenomic works has historically
been driven entirely by the availability of sequence-grade tissues, rather than by
phylogenetic representation, given that the internal phylogenetic structure of this order
has never been fathomed (Borner et al. 2014; Sharma et al. 2014; Ballesteros and
Sharma 2019; Arribas et al. 2020; Ballesteros et al. 2022; Ban et al. 2022). It is possible
that this oversight may have underlain their known predilection for topological instability
in some phylogenomic datasets (Sharma et al. 2014; Ballesteros and Sharma 2019;
Ballesteros et al. 2019).
To redress this basic gap in our understanding of Solifugae, we generated the first
higher-level phylogeny of the group, leveraging natural history collections worldwide to
sample all extant families and reconcile a dated phylogeny against the fossil record. We
obtained a robust backbone tree topology, with nearly all interfamilial nodes well-
supported and recovered across an array of occupancy thresholds with support. Our
results show that most solifuge families represent cohesive groups, with many families
exhibiting high fidelity to specific biogeographic terranes. Curiously, we recovered the
North American family Eremobatidae as the sister group of Gylippidae, which is
restricted to the Old World (Central Asia and South Africa), with these two taxa in turn
sister group to a large clade of Old World families (Galeodidae, Karschiidae, and
Rhagodidae). This result was unanticipated because gylippids were previously
considered part of Karschiidae (Roewer 1933), and because a North American sister
group of this family implies a markedly disjunct distribution. However, this relationship is
consistent with shared traits of cheliceral architecture and dentition in these two families
(Bird et al. 2015).
Paralleling this outcome, the other groups of New World solifuges were also recovered
as nested within a clade of Old World taxa (Ceromidae, Solpugidae, Hexisopodidae,
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and Old World daesiids). Our analyses consistently recovered a clade formed by the
South American family Mummuciidae, the New World family Ammotrechidae, and the
South American subset of the paraphyletic group Daesiidae (the “Daesiidae III” clade).
Within each, we additionally found close relationships between geographically
proximate subtaxa. These results suggest a prominent phylogenetic signature in
solifuge biogeographic distributions, and across varying depths of the phylogeny.
Three relationships exhibited instability across analyses and invite further scrutiny.
First, the fossorial group Hexisopodidae (commonly, “mole solifuges”), from southern
Africa, exhibited some topological instability being recovered as either the sister group
of Solpugidae in a minority of analyses, or with a clade formed by Daesiidae +
Mummuciidae + Ammotrechidae. Both placements are partially supported by available
morphological data. Specifically, analyses of solifuge sperm ultrastructure previously
showed that hexisopodids and solpugids share similarities in the fine structure of
spermatozoa, such as a conical acrosomal vacuole that is located within the chromatin
body (Klann et al. 2011); an older work had also suggested that hexisopodids may
constitute a derived subtaxon of Solpugidae (Hewitt 1919). But hexisopodids also
exhibit digitiform protuberances of membranes and putative deposits of stored
glycogen, which are similarly observed in Daesiidae and Ammotrechidae, in addition to
Solpugidae (Klann et al. 2011). These data strongly accord with the recovery of these
four families in a clade across our analyses, but we add the caveat that morphological
data for solifuge sperm ultrastructure remain fragmentary; characteristics of the
spermatozoa have been surveyed only in seven of the 12 families to date (Klann et al.
2009, 2011). In particular, data on the sperm ultrastructure of Mummuciidae, a member
of this group, are not presently available.
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The second relationship that exhibited instability was the placement of the sole
exemplar of Melanoblossidae; a family with a disjunct distribution that encompasses
southern Africa and Southeast Asia. A minority of analyses recovered Melanoblossia as
the sister group of the Old World Daesiidae, whereas the majority recovered this
terminal as derived within the Old World daesiids. As Melanoblossidae was previously
considered a subfamily of Daesiidae (Roewer 1933), we cannot rule out a nested
placement of this taxon. As with the hexisopodids, this topological instability across
analyses stems from limitations in locus representation for Melanoblossia, which
confers a high proportion of missing data for these terminals in the phylogenetic
matrices we analyzed. Future investigations of hexisopodid and melanoblossid
placement must emphasize deeper sampling and sequence coverage for these two
taxa.
A final relationship that exhibited instability across analyses was the placement of
Mummuciidae. While mummuciids were typically recovered as nested within
Ammotrechidae, we did recover this clade as the sister group of the ammotrechids in a
minority of analyses (albeit without support). As with the previously discussed case,
mummuciids were previously a subtaxon of Ammotrechidae, and thus a nested
placement of this putative family would not be unprecedented (Roewer, 1933). Given
the complexity of ammotrechid subfamilies (a subset of which were sampled here), we
submit that future systematic revisions of the South American solifuges must consider
mummuciids as potentially derived members of Ammotrechidae.
With regard to biogeography, the chronological sequence of Pangean fragmentation is
well-documented, and numerous cases of biotic distributions and divergence time
estimates have been shown to retain the signature of supercontinental breakup (e.g.,
Sanmartín and Ronquist 2004; Giribet et al. 2012; Mao et al. 2012; Murienne et al.
2013b). Our biogeographic analysis revealed the general pattern that the distribution of
most solifuge clades is delimited by single biogeographic regions. Ancestral area
optimizations in the internal nodes and the distribution of extant species (e.g., near-
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absence on oceanic and Darwinian islands) suggest that solifuges are habitat
specialists adapted to specific environments, being largely confined to xeric habitats.
Like many arachnid orders, the crown group of Solifugae dates to the Carboniferous or
earlier, as reflected by its fragmentary fossil record (World Solifugae Catalog 2022;
Dunlop 2010). Most solifuge families and genera are relatively young, diversifying soon
after the Cretaceous-Paleogene extinction event, particularly in Clade I. While this may
reflect an artifact of limited sampling, one possibility for the observation of relatively
young ages of family-level clades is that the recent aridification and the opening of
comparatively young deserts may have opened new ecological niches for Solifugae,
driving the diversification of this arid-adapted arachnid group by the mid-Cenozoic
(Zhang et al. 2014; Zheng et al. 2015; Cushing et al. 2015). Their absence on dry
continental landmasses such as Australia may be attributable to the recent timing of
aridification of the Sahul Shelf, as well as the mesic and cooler past conditions of the
Australian interior (Byrne et al. 2011). However, assessing this hypothesis using
parametric tests requires extensive sampling of extant diversity, in tandem with
consideration of variance in the ages of deserts worldwide. Targeted approaches like
UCE sequencing offer the promise of renewed utility of natural history collections and
revitalized prospects for hypothesis testing with poorly studied taxa.
We emphasize that various subtaxa were not included in this study, which is aimed
specifically at higher-level relationships between the families. Given the fidelity we
observed between solifuges and the terranes that they inhabit, we anticipate that future
sampling efforts will identify additional cases of non-monophyletic taxa from regions not
represented in this study (e.g., the Indian subcontinent; eastern China), which will
require intensive rounds of systematic revision under a phylogenomics lens. Such
genomics-driven revisionary efforts have demonstrated marked success and efficiency
in modernizing the classification and evolutionary analysis of comparably diverse
groups, such as scorpions and harvestmen (Sharma et al. 2015; Derkarabetian et al.
2018; Benavides et al. 2019b; Santibáñez-López et al. 2019, 2020).
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Conclusions
The phylogenetic tree topopology presented herein bookends a ca. 20-year gap of
available higher-level phylogenies for the orders of Chelicerata (Harvey 2002). The
relationships inferred herein are anticipated to aid taxonomic revisionary efforts, and
revitalize morphological comparative studies and biogeographic efforts within this
understudied group. Future investigations of solifuge evolutionary history must
emphasize the role of global perturbations of the past and the downstream effects of
climatic cycles on diversification and range expansion of these cryptic, long-neglected
arthropods.
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15
Materials and Methods
Species sampling
Specimens sequenced for this study were collected from field sites as part of our recent
collecting campaigns, as well as contributed by collections of the Museo Argentino de
Ciencias Naturales “Bernardino Rivadavia”, Buenos Aires, Argentina (MACN); The
National Natural History Collections, The Hebrew University, Jerusalem, Israel (NNHC);
the Denver Museum of Nature & Science, Colorado, United States (DMNS); the
National Collection of Arachnida, Agricultural Research Council, South Africa (ARC-
PCP), and the Zoological Museum Hamburg (ZMH). Collecting permits for study taxa
were issues to different laboratories over several years; permitting data are available
upon request from the authors. For de novo sequencing of UCEs, sampled exemplars
of each of the 12 described extant families, as follows: 18 Ammotrechidae, six
Ceromidae, 15 Daesiidae, five Eremobatidae, nine Galeodidae, two Gylippidae, four
Hexisopodidae, eight Karschiidae, five Melanoblossidae, six Mummuciidae, ten
Rhagodidae, and 25 Solpugidae. To this dataset, we added UCE loci from five
published solifuge transcriptomes, comprising one Ammotrechidae, two Eremobatidae
and two Galeodidae
Outgroup sampling was influenced by previous works that have inferred various
possible placements of Solifugae in the chelicerate tree of life, such as a sister group to
Acariformes and Palpigradi, or part of a clade with Riniculei, Opiliones, and Xiphosura
(Pepato et al. 2010; Sharma et al. 2014; Ballesteros and Sharma 2019; Ballesteros et
al. 2019, 2022). Given this instability across studies, we sampled 2-3 terminals of every
extant chelicerate order, prioritizing the sampling of basal splits in each outgroup order.
Outgroup datasets were drawn from published transcriptomes and from our previous
UCE assemblies that were captured with the same probe set. All tree topologies were
rooted with Pycnogonida.
Ultraconserved element sequencing and phylogenomic analyses
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For newly sequenced specimens, 1-4 legs or tissue teased from a single chelicera from
single specimens were used for DNA extractions using the DNeasy™ Blood and Tissue
kit and the QIAamp DNA Mini kit (Qiagen Inc., Valencia, CA). Libraries were prepared
and enriched following protocols outlined by (Kulkarni et al. 2020) and Miranda et al.
(2022). All pools were enriched with the Spider2Kv1 probe set (Kulkarni et al. 2020)
following the myBaits protocol 4.01 (Arbor Biosciences). Sequencing was performed on
an Illumina NovoSeq platform. Assembly, alignment, trimming and concatenation of
data were performed using the PHYLUCE pipeline (publicly available at
https://phyluce.readthedocs.io/en/latest/). UCE contigs were extracted using the
Spider2Kv1 probe set (Kulkarni et al. 2020) to target 2,021 UCE loci. To augment the
UCE dataset with RNASeq datasets, we followed the assembly, sanitation, reading
frame detection, and UCE retrieval pipeline outlined by Kulkarni et al. (2021). Homology
screening was performed by employing liberal (65%) and stringent (80%) probe-to-
library identity and coverage mapping thresholds as suggested by Bossert & Danforth
(2018).
Phylogenomic analyses
The assembly, alignment, trimming and concatenation of data were done using the
PHYLUCE pipeline (publicly available at https://phyluce.readthedocs.io/en/latest/). We
assessed the sensitivity to data completeness by applying gene occupancy with
successive increments of 5% (10% onwards) to where the locus count dropped below
50 UCE loci.
Orthologous and duplicate loci were screened with the minimum identity and coverage
of 65%. Phylogenetic analyses were performed on the unpartitioned nucleotide data
using IQ-TREE (Nguyen et al. 2015) version 2. Model selection was allowed for each
unpartitioned dataset using the TEST function (Kalyaanamoorthy et al. 2018, Hoang et
al. 2018). Nodal support was estimated via 2,000 ultrafast bootstrap replicates (Hoang
et al. 2018) with 10,000 iterations. To reduce the risk of overestimating branch support
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with ultrafast bootstrap due to model violations, we appended the command -bnni. With
this command, the ultrafast bootstrap optimizes each bootstrap tree using a hill-climbing
nearest neighbor interchange (NNI) search based on the corresponding bootstrap
alignment (Hoang et al. 2018).
GC-content
GC content can influence the phylogenetic relationships reconstructed using genome
scale data (Benjamini and Speed 2012). To address this, we computed GC content for
each taxon in the concatenated alignment using a custom script paired with BBMap
(https://github.com/BioInfoTools/BBMap).
Phylogenomic dating
As no molecular phylogeny of the order exists and the solifuge fossil record is sparse,
the timeframe of solifuge diversification is effectively unknown. To provide a temporal
context to the divergences we inferred, we performed divergence time estimation using
a least-squares approach, LSD2 method (To et al. 2016) which uses a least-squares
approach based on a Gaussian model and is robust to uncorrelated violations of the
molecular clock. LSD2 requires at least one fixed date, so we used an absolute
calibration of 314.6 Ma for the crown Orthosterni fossil, Compsoscorpius buthiformis.
We optimized the fossil information-based calibrations on the tree topology inferred from
the 25% occupancy data set for the basis for divergence time estimation, implementing
uniform node age priors to accommodate the scarcity of terrestrial chelicerate fossils.
The root age was set to a range of 550–600 Mya, following Wolfe et al. (2016). The
crown age of Solifugae was constrained using a minimum age bound of 305 Ma, based
on the Carboniferous fossil Prosolpuga carbonaria (Petrunkevitch, 1913). The stem age
of Ceromidae was constrained using a minimum age bound of 115 Ma, based on the
Cretaceous fossil Cratosolpuga wunderlichi (Selden and Shear 2016). The recently
described Burmese amber fossil Cushingia ellenbergeri was not included for calibration,
characters of this species potentially accord with a melanoblossid, a gylippid, or a
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18
rhagodid identity, which precludes its use for calibrating a specific node (Dunlop et al.
2015; Bartel et al. 2016). Outgroup nodes were calibrated using the oldest
unambiguous fossils representing those clades.
Ancestral area reconstruction
We reconstructed ancestral areas on internal nodes of the dated preferred tree and the
combined tree using the package BioGeoBEARS (Matze, 2013) implemented in RASP
4.0 (Yu et al., 2020). Each of the terminals was assigned to one of the following
biogeographic regions: Turanian, African tropics, Neotropical, Nearctic, Central Chile-
Patagonia and Mediterranean. We chose this scheme of area coding based on the
distribution of the extant solifuges and following commonly used area definitions from
the literature. Some taxa such as ammotrechids and eremobatids are distributed in the
United States and Mexico. For these taxa, we coded the area as Nearctic following the
delimitation of this region by Escalante et al. (2010). Additionally, to assess the
influence of area coding, we alternatively coded areas by continents (corresponding to
their geological plate); and more coarsely, as either Laurasia or Gondwana, based on
the geological origin of those areas.
Acknowledgements
We are grateful to Nuria Macias (Departamento de Zoología de la Universidad de La
Laguna) for providing a specimen of Eusimonia wunderlichi from the Canary Islands,
and to Marc Domènech and Miquel Arnedo (University of Barcelona) for providing a
specimen of Gluvia dorsalis from Spain. Photographs of live specimens were kindly
provided by Igor Armiach Steinpress and Shlomi Aharon. Sequencing was performed at
the Biotechnology Center of the University of Wisconsin-Madison.
Funding
This project was supported by Binational Science Foundation grant no. BSF-2019216 to
EGR and PPS; and National Science Foundation grant no. DEB-1754587 to PEC. SSK,
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GG, and PPS were additionally supported by National Science Foundation NSF grant
no. IOS-2016141 to PPS.
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Figure 1. Live habitus of Solifugae. (A) Adult female of Eremobates (Eremobatidae)
from Arizona, US. (B) Brooding female of Galeodes (Galeodidae) over a clutch of
hatchlings, from Israel. (C) Adult female of Mummucia (Mummuciidae) from Chile. (D)
Adult male of Procleobis patagonicus (Ammotrechidae) from Argentina. (E) Adult female
of Blossia (Daesiidae) from Israel. (F) An unidentified Daesiidae from Israel. (G) An
unidentified Rhagodidae from Israel. (H) An actively burrowing Chelypus
(Hexisopodidae) from Namibia. Photographs: G. Giribet (A); I. Armiach (B, E, F, G); H.
Iuri and A. Ojanguren-Affilastro (C, D); S. Aharon (H).
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Figure 2. A maximum likelihood phylogeny of Chelicerata with all Solifugae family
representatives using the 25% occupancy data set of ultraconserved elements.
Figure 3. A comparison of interfamilial phylogenetic relationships of Solifugae
compared across results of all analytical treatments. A. A maximum likelihood based
phylogeny using the 25% occupancy data set (same as figure 2). B. Corroboration of
relationships across different data sets at each node is shown in color palettes,
alternative relationships are shown in white and NA indicates that either the family
represented a single taxon or was not included in the analysis.
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Figure 4. Fossil-calibrated dated phylogeny of Solifugae. Stars indicate fossil
placements used for calibrating node ages. Outgroups removed for clarity.
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Figure 5. A. Biogeographic hypothesis obtained from the Dispersal-Extinction-
Cladogenesis + jump parameter model of RASP analyses on the fossil-calibrated LSD2
analysis using the 25% occupancy data set. The dotted line in red indicates the
Cretaceous Triassic boundary. B. Graphics for Pangean breakup from Scotese, C.R.,
2016. PALEOMAP Project, http://www.earthbyte.org/paleomap--
paleoatlas--
for-
-
gplates/. Ancestral areas optimized for each family and higher-level nodes are
marked at the respective MRCA nodes. Red arrows indicate dispersal implied by the
DEC+j model whereas the colored arrowheads indicate alternative vicariance events
implied by the exclusion of the jump parameter.
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