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\begin{document}

\title{The phylogeography and diversification of an endemic trapdoor spider
genus, Stasimopus Simon 1892 (Araneae, Mygalomorphae, Stasimopidae) in
the Karoo, South Africa}



\author[1]{Shannon Brandt}%
\author[1]{Catherine Sole}%
\author[2]{Robin Lyle}%
\affil[1]{University of Pretoria}%
\affil[2]{Agricultural Research Council}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Aim: The genus Stasimopus is endemic to South Africa but has never
undergone a phylogeographic review. This study aims to unravel the
phylogeographic patterns and history of the many Stasimopus species
which occur in the greater Karoo region. Location: The Karoo region,
South Africa Time period: 2017-2018 Major taxa studied: Cork-lid
trapdoor spider, Stasimopus (15 species). Methods: A fossil-calibrated
phylogeny was produced based on three gene regions (CO1, 16S and EF-1g\selectlanguage{english})
for Stasimopus specimens collected in the Karoo region, to infer dates
of origin and diversification. Demographic analyses were performed on
species with sufficient sample sizes (\textgreater{}4). Haplotype
networks were constructed for each gene region and plotted on a map to
infer phylogeographic patterns. Lastly, Mantel tests were performed to
test for isolation by distance. Results: It was found that 15 species
occur in the Karoo and that the genus radiation in the area is in the
early Paleocene. Most diversification occurred between the late Eocene
and the Miocene. Several species show signals of demographic expansions.
Isolation by distance was detected, but only with a slight correlation.
Main conclusions: It is apparent that aridification has played a vital
role in the diversification of the genus in the Karoo region. This is a
shared biogeographic influence between the mygalomorph fauna of the
Karoo and arid region of western Australia. Stasimopus has radiated from
the late Eocene and through the Miocene resulting in 15 extant species
in the region. The Tankwa Karoo has been identified as a possible
Pleistocene glacial cycle refugia for the species S. leipoldti. Many of
the species in the Karoo are short range endemics, making them of high
conservation concern. This study provided vital information as the Karoo
is undergoing further desertification due to factors such as climate
change, which may affect the future of short-range endemic spiders.%
\end{abstract}%



\sloppy


\textbf{The phylogeography and diversification of an endemic trapdoor
spider genus, \emph{Stasimopus}Simon 1892 (Araneae, Mygalomorphae,
Stasimopidae) in the Karoo, South Africa}

\textbf{Radiation of \emph{Stasimopus} in the Karoo}

\textbf{Abstract}

\textbf{Aim:} The genus Stasimopus is endemic to South Africa but has
never undergone a phylogeographic review. This study aims to unravel the
phylogeographic patterns and history of the many Stasimopus species
which occur in the greater Karoo region.

\textbf{Location:} The Karoo region, South Africa

\textbf{Time period:} 2017-2018

\textbf{Major taxa studied:} Cork-lid trapdoor spider,
\emph{Stasimopus}(15 species).

\textbf{Methods:} A fossil-calibrated phylogeny was produced based on
three gene regions (CO1, 16S and EF-1g\selectlanguage{english}) for Stasimopus specimens
collected in the Karoo region, to infer dates of origin and
diversification. Demographic analyses were performed on species with
sufficient sample sizes (\textgreater{}4). Haplotype networks were
constructed for each gene region and plotted on a map to infer
phylogeographic patterns. Lastly, Mantel tests were performed to test
for isolation by distance.

\textbf{Results:} It was found that 15 species occur in the Karoo and
that the genus radiation in the area is in the early Paleocene. Most
diversification occurred between the late Eocene and the Miocene.
Several species show signals of demographic expansions. Isolation by
distance was detected, but only with a slight correlation.

\textbf{Main conclusions:} It is apparent that aridification has played
a vital role in the diversification of the genus in the Karoo region.
This is a shared biogeographic influence between the mygalomorph fauna
of the Karoo and arid region of western Australia. Stasimopus has
radiated from the late Eocene and through the Miocene resulting in 15
extant species in the region. The Tankwa Karoo has been identified as a
possible Pleistocene glacial cycle refugia for the species S. leipoldti.
Many of the species in the Karoo are short range endemics, making them
of high conservation concern. This study provided vital information as
the Karoo is undergoing further desertification due to factors such as
climate change, which may affect the future of short-range endemic
spiders.

\textbf{Keywords}

\textbf{divergence dating, Karoo, Mygalomorphae, phylogeography, South
Africa, \emph{Stasimopus}}

\textbf{Introduction}

The Karoo region of South Africa has long been neglected in studies due
to the assumption of low biodiversity in semi-arid regions . This began
to change in 2015 when a Strategic Environmental Assessment (SEA) was
undertaken to produce a report on the state of biodiversity in the
region to assist governmental policy-makers in making informed decisions
about land use change in the area through the Karoo BioGaps project .
This became imperative as in previous years pressure for land use change
increased due to economic incentives such as mining, farming, renewable
energy, intensive shale gas exploration and the Square Kilometre Array
(SKA) . To date there is still however, few studies on the faunal
diversity in the area .

The Karoo region of South Africa has a geologically rich history. The
Karoo basin of South Africa in combination with four other basins
globally form the Gondwanan foreland basins which were formed by
collisions and terrain accretion tectonics along Gondwana's southern
edge . The Karoo basin sedimentation dates from the late Carboniferous
to the middle Jurassic (323 -- 174 MYA) . During this time the
sedimentation recorded a gradual change from a glacial environment to an
arid desert, as South Africa changed in latitude from the polar region
to the tropics . The Karoo basin is the only basin globally to record
this 200 MY change in its sedimentation . It spans 700,000
km\textsuperscript{2}, covering 60\% of South Africa's land surface .
Only the South Western portion of this is the modern-day Karoo region.
Due to the geological history of the basin, it is now of large economic
value due to expansive coal deposits .

The modern-day Karoo region of South Africa is an arid/semi-arid area
that spans one third of the land surface of South Africa . The Karoo
comprises two separate biomes, namely Nama Karoo and Succulent Karoo,
which are distinguished by climate and vegetation . The Nama Karoo is on
the central plateau in the western portion of the country . It
experiences unreliable summer rainfall with periods of prolonged drought
. The vegetation is dominated by dwarf shrub vegetation which are
xeromorphic as well as succulents in the west and grasses in the east .
This is the second largest biome in South Africa covering 260,295
km\textsuperscript{2} . Due to past landuse practices approximately 60\%
of the biome has moderate to severe degredation of soils and vegetation
. Only 2\% of the entire Nama Karoo biome is under conservation . The
Succulent Karoo covers a belt from L\selectlanguage{ngerman}üderitz (Namibia) through
Namaqualand and includes smaller areas of Hantam, Tanqua, Roggeveld and
the little Karoo . The biome experiences reliable winter rainfall
(100-200 mm anually) which aids in the sustainability of vegetation .
The vegetation is dominated by succulent plants and following good rains
by annuals (Asteraceae and Brassicaceae) . This biome is the forth
largest in South Africa and covers 87,001 km\textsuperscript{2} . The
succulent Karoo is bordered by the Cape Fold mountains, which caused
isolation leading to high levels of endemisim and species richness of
many taxa including hopliniid beetles, aculeate Hymenoptera, reptiles
and arachnids . The succulent Karoo is also the world's only entirely
arid region diversity hotspot . Despite high levels of endemisim and
being a biodiversity hotspot, only 8\% of the biome is conserved .

Stasimopidae is a family of trapdoor spiders endemic to Southern Africa
. The family consists of one genus, \emph{Stasimopus} and 56 described
species . These spiders have life history traits which make it easy to
detect population structuring and dispersal events increasing the
likelihood of inferring phylogeographic events. Mygalomorphs are
exceptionally long lived for spiders, reportedly living to 43 years old
. Most mygalomorph spiders live in retreats comprising vertical burrows
or chambers under rocks or on trees which are lined with silk . The
females show great fidelity to their burrows, hunting from burrow
entrances . The males are nomadic, wandering to locate females to mate
with and are thus responsible for nuclear gene flow between populations.
The offspring, once mature enough, will leave the mothers burrow but
only move a few meters before making a new burrow to live in . This
combination of behaviours leads to extensive population structuring and
isolated geographic locations for the populations (Rix et al., 2017).
These characteristics are often associated with species which are
classified as `short range endemics', which if found to be true for the
species of the genus, may make them of conservation
concern.\emph{Stasimopus} and other mygalomorph spiders are model
species for phylogeographic studies due to these factors . Performing
this study on the \emph{Stasimopus} genus, could possibly serve as a
proxy for other mygalomorph spiders living under similar environmental
conditions.

Several phylogeographic studies have been performed on Mygalomorphae
species, but none have to date been performed in South Africa, nor on
the \emph{Stasimopus} genus . Phylogeographic inference is of immense
importance to conservation as there is the possibility of defining areas
for conservation based on the evolutionary potential of various taxa .
This is vital in light of the sensitivity of the Karoo ecosystem, the
life history of \emph{Stasimopus} and the drastic land-use and climate
change predicted for the area.

The aims of this study are threefold; first, to create a dated phylogeny
for the \emph{Stasimopus} species occurring in the Karoo region using
CO1, 16S and EF-1\selectlanguage{english}g genes. This will be used to understand the
diversification of the genus over geological time. Second, using
haplotype networks to understand the distribution of genetic diversity
in the landscape. Lastly, evaluating the phylogeographic pattern
underlying the phylogeny and assessing the potential drivers thereof.

\textbf{Materials and Methods}

\emph{Taxon sampling}

\emph{Stasimopus} specimens were collected from part of the Great Karoo,
South Africa. The area is within the demarcated area for potential shale
gas fracking. The boundary of the area is set approximately by the
following coordinates: -30.88688, 26.29295 and -33.03079, 20.01661
(Figure 1). Sites were selected by the Karoo BioGaps team to cover the
range of environmental conditions present in the region and within each
site drainage line habitats were targeted for sampling as they are
favoured by trapdoor spiders. \emph{Stasimopus} specimens were collected
at 55 sites. At each site four people spent one-hour, thereby four
man-hours soil scraping. Soil scraping involves removing the top layer
of soil to expose burrow entrances. Once entrances were exposed, the
burrows were excavated using a hand shovel, taking care to not injure
the specimens. This methodology was effective for collecting female and
juvenile specimens. In order to collect male specimens, road cruising
was done after rain in the evenings. This involves slowly driving on
isolated roads with torches to find males which are crossing in search
of potential mates. All specimens were preserved in a solution of cold
alcohol and kept in an ice box. Specimens were later preserved in 80\%
ethanol in glass polytop vials for long term storage. All locality
information is available in Table S1. All newly collected material was
deposited in the National Collection of Arachnida (NCA) of the
Agricultural Research Council (ARC), Roodeplaat, South Africa.

\emph{Sequence data generation}

DNA extraction and sequencing are as stated in Brandt et al. (2023). All
sequences generated were assembled in CLC Bio Main Workbench Version 6.9
(http://www.clcbio.com). All gene regions were submitted to GenBank and
the accession numbers are recorded in Table S1. The CO1, 16S and EF-1g
\selectlanguage{english}datasets were concatenated using FASconCAT v1.11 (K\selectlanguage{ngerman}ück and Meusemann,
2010). The edited sequences for each gene region as well as the
concatenated dataset, were aligned using MAFFT online (Katoh, 2005;
Katoh \& Toh, 2008). The `Auto' strategy for alignment was used in
MAFFT, this inspects the direction of the sequences and adjust the
alignment in correlation to the first sequence.

\emph{Divergence dating}

Dating the phylogeny was done using BEAST v1.8.4 . All identical
sequences were removed from the analysis. In order to determine the most
appropriate fossil calibration points, numerous combinations were tested
varying the fossil calibrations used. The final fossil calibration
points used were as follows: the Hexathelidae fossil,\emph{Rosamygale
grauvogely} (Gresa-Voltzia formation, France, Triassic) .
\emph{Rosamygale grauvogely} is thus, the first mygalomorph appearance
in the fossil record, dating to 250 -- 240 MYA . The Nemesiidae
fossil,\emph{Cretamygale chasei} (Isle of Wight, Cretaceous) , two
Ctenizidae/Halonoproctidae fossils, \emph{Ummidia damzeni} and \emph{U.
malonowskii} (both from Baltic amber, Palaeogene) . Lastly, a fossil
from the family Cyrtaucheniidae, \emph{Bolostromus destructus}(Dominican
amber, Neogene) . Each family was represented by available sequence data
from GenBank for 16S rDNA, cytochrome oxidase 1 (CO1) and elongation
factor 1 gamma (EF-1\selectlanguage{english}g) (Table S5). Each calibration point was set by
setting a hard minimum bound (the youngest possible age of the fossil
based on the deposit in which it was found) and a soft upper bound (the
oldest possible age).

The nucleotide substitution rate was determined using jModelTest v2.1.7
. This was done for each gene region and the best model to account for
varying base pair substitution rates was selected based on the Bayesian
information criterion (BIC) . A summary of the number of samples for
each gene region, sequence length and substitution model are given in
Table S6. This was used to set the prior in BEAUTi v1.8.4 . Other priors
set included, the rate of molecular evolution to a relaxed clock with a
lognormal distribution (this allows mutational rates to vary over the
tree) . As the dataset includes intraspecific and interspecific sampling
the study follows the protocol of by testing four different tree priors.
All analyses were thus repeated using `Speciation: Birth-Death
Incomplete Sampling', `Speciation: Birth-Death', `Speciation: Yule
Process' and `Coalescent: Constant Size' at tree priors. This allowed
testing at both the population level of diversity and species level
diversity. An uncorrelated relaxed clock was used, as this allows for
each branch of the phylogeny to have a different evolutionary rate .
BEAST was then run for 200,000,000 (250,000,000 for the yule model)
generations and sampled every 5000 generations. This was repeated twice
for each analysis to ensure convergence. BEAST was run in conjunction
with BEAGLE .

Tracer v1.7.1 was used to confirm convergence . This was tested by
checking the log file for each BEAST run and ensuring the ESS values
were above 200. The individual runs were then compiled to ensure a
normal distribution. Log Combiner v1.8.4 ~was used to combine the
multiple tree files into one file. The subsampling number was set to
50,000 and the burn-in for each tree to 50,000,000.

The tree files viewed, annotated and edited in FigTree .

\emph{Demography analyses}

DNA sequence polymorphism (DnaSP) v6.12.03 was used to gather baseline
statistics of gene region for each species with more than four
representatives. Ambiguous nucleotides were removed, and all other
settings left on default. The basic statistics recorded were as follows:
nucleotide diversity (\selectlanguage{greek}π\selectlanguage{english}), gene diversity (H\textsubscript{d}), number of
haplotypes (H), mean number of pairwise differences (k) and number of
segregating sites (S). Tajima's test and Fu and Li's tests were also run
. Tajima's D compares the number of segregating sites (S) and the mean
pairwise differences between different sequences . Fu and Li's D* is
similar but compares the total amount of nucleotide variations to the
number of singleton mutations, this was done without an outgroup . Both
tests are statistical tests of neutrality and are used to determine
historic population demographic changes .

\emph{Haplotype networks}

DnaSP was also used to prepare the sequence data for input into Network
v5.0.0.0 in order to construct haplotype networks. The 16S data was
exported in Rdf format with gaps/missing data not considered, whereas
the coding regions (CO1 and EF-1g\selectlanguage{english}) were exported with gaps considered.
In order to accurately estimate the relationship between closely related
\emph{Stasimopus}individuals of the same species (which have not yet
undergone speciation), a haplotype network was constructed in Network.
The Network software uses the method of Maximum Parsimony to select the
shortest and least complex tree. The haplotype network was constructed
using the median-joining (MJ) network algorithm. This allows for
multistate data (A, T, G, C or N at any nucleotide position) . All
settings for network calculation were left on default. The
post-processing option of Maximum parsimony was used in order to remove
all unnecessary median vectors.

Pie charts of haplotypes were plotted onto a map of the Karoo region
using the coordinates of each locality. This allowed for the
visualisation of the different haplotypes in geographic space.

\emph{Mantel test and isolation by distance}

A mantel test was performed to determine if geographic distance and
genetic distance are correlated. This determines if there is isolation
by distance. A distance matrix for the sampling localities was
constructed by determining the distance between each set of coordinates
using the online tool, Movable Type Scripts: Calculate distance, bearing
and more between Latitude/Longitude points (Available at:
https://www.movable-type.co.uk/scripts/latlong.html) . This tool uses
the haversine formula to calculate the distance between coordinate pairs
. A distance matrix was constructed from the pairwise genetic distance
for the CO1 dataset (as this is the most complete dataset) using Mega 7
.

The Mantel test was conducted for two different datasets in R (R Core
Team, 2017), using the packages `Ade4' (Dray et al., 2018) and `vegan'
(Oksanen et al., 2019) and were both run for 10,000 permutations. First
was to determine if there is genetic isolation for each locality, this
thus included the 48 localities for which CO1 data is available and one
representative sequence per locality, in their respective distance
matrix. The second test was to test if there is isolation by distance
across the various species, for this, localities which were at the edges
of the species distribution were chosen across the various species. The
specimens used are available in Table S7.

\textbf{Results}

\emph{Divergence dating}

The dated phylogeny for the \emph{Stasimopus} genus in the Karoo can be
seen in Figure 2. Samples indicated in red text (and the grey bar on the
right) could not be identified to species level as they are juveniles.
Divergence estimates indicate that the genus \emph{Stasimopus} in the
Karoo dates to between 48.30 - 84.15 MYA, towards the beginning of the
Paleocene under the birth-death speciation model (Figure 2, node i).
This range is shared by the birth-death incomplete and coalescent models
(Figures S1 and S2). The yule speciation model gives older node ages
across the entire phylogeny (Figure S3, Table 1). Due to the consistent
dating of the birth-death, birth-death incomplete and coalescent models,
the discussion will revolve around these dates.

The phylogeny is divided into 11 clades for the ease of discussion
(labelled A - K). Clade A is the oldest clade across all models. This
clade is well supported and consists of a species complex of \emph{S.
erythrognathus} and \emph{S. karooensis} as well as a single individual
representing \emph{S. theaei.} This clade is dated to 24.13 -- 50.25 MYA
(Figure 2, node ii). The age of the species complex is younger 9.36 -
22.35 MYA (Figure 2, node iii), the inclusion of \emph{S. theaei} and
the unidentified samples make the clade significantly older. Clade B is
a well-supported monophyletic clade, consisting of \emph{S. astutus}
and\emph{S. finni.} The species \emph{S. astutus} is young from the
Miocene (6.89- 16.46 MYA) (Figure 2, node iv), but Clade B is dated
between 22.69 - 50.78 MYA due to the inclusion of \emph{S. finni}
(Figure 2, node v). Clade C is dated within the Oligocene, the
individual species cannot be accurately dated due to small sample sizes.
\emph{Stasimopus dylani} (Clade D) (Figure 2, node vi), \emph{S.
venterstadensis} (Clade I) (Figure 2, node iiv) and \emph{S.
unispinosus} (Clade H) (Figure 2, node iiiv) are all dated to the
Miocene. Clade F (\emph{S. maraisi} ) is one of the older species dated
between 16.2 -- 39.32 MYA (Figure 2, node ix), followed by clade G
(\emph{S. hamartia} ) between 22.53 -- 43.56 MYA (Figure 2, node x).
\emph{Stasimopus leipoldti} (Clade K) is very recently diverged in the
Pleistocene (Figure 2, node xi). This may however, be inaccurate due to
the small sample size and that the samples are from the same locality.
Clades E and J are comprised of only unidentifiable juveniles. It is
however apparent that these specimens likely comprise separate species.

\emph{Demographic history}

The haplotypes and demographic results for CO1 are presented in Figure 3
and Table 2 and the data for 16S and EF-1g \selectlanguage{english}in Figures S4, S5 and Tables
S8, S9, respectively. The haplotype networks are largely in agreement
with the phylogeny, except for the placement of the \emph{S.
venterstadensis} (Clade I, light green) individuals in the CO1 network
(Figure 3) which are split and group with \emph{S. maraisi} (Clade F,
dark blue) and \emph{S. astutus} (Clade B, coral) separately. The
species complex of clade A (Figure 2) appears to be present across all
three haplotype networks. The networks reveal that there is a higher
degree of genetic differentiation in the CO1 gene within species than
the other two gene regions, this is apparent from the higher number of
mutational steps between haplotypes. EF-1g \selectlanguage{english}consistently has the least
number of mutational steps between haplotypes within species. This is
further substantiated by the differences in \emph{k, S} and
\selectlanguage{greek}\emph{π}\selectlanguage{english}values across the three gene regions for each species (Table 2,
Tables S8 and S9). This should however be interpreted with caution as
all sample sizes (except for \emph{S. maraisi} ) are quite low.

The haplotype diversity is high across all species considered for the
CO1 gene region (Table 2). \emph{Stasimopus unispinosus} has a lower
haplotype diversity for the 16S and EF-1g \selectlanguage{english}regions, both of 0.6 (Tables
S4 and S5). Nucleotide diversity is relatively low across species and
gene regions (\textgreater{}0.1) (Table 2, S8 and S9).

In the EF-1g \selectlanguage{english}network (Figure S5) the \emph{S. maraisi} network shows a
clear expansion pattern, but this is not shown in the other two gene
regions. This expansion of \emph{S. maraisi} for the EF-1g \selectlanguage{english}gene region
is further proven by the significant negative result of the Tajima's D
(-1.825*) and Fu \& Li's D* (-3.140*) statistics. The only other
significant result is for \emph{S. hamartia} for 16S, where Fu \& Li's
D* is significant (1.418).

There is clear phylogeographic structuring across all three gene
regions. Most species appear to be isolated to one or a few locations,
except for \emph{S. maraisi} which is widespread across the
region.\emph{Stasimopus leipoldti} was only found in one location in the
Tankwa Karoo, but the type locality for the species is Clanwilliam
(Western Cape), over 150 km away. A similar trend is seen for \emph{S.
erythrognathus} which was in found in the eastern Karoo, but the type
locality is Worcester (Western cape), 700 km away.

There are many species occurring sympatrically at multiple locations.
These include \emph{S. maraisi} and \emph{S. hamartia} ; \emph{S.
maraisi} and \emph{S. dylani} ; \emph{S. theaei} and \emph{S. finni}
;\emph{S. erythrognathus} and \emph{S. karooensis} ; \emph{S. astutus}
,\emph{S. mandelai} and \emph{S. dylani} ; \emph{S. maraisi} and Clade
J. Clade J likely comprises a new species based on the degree of genetic
variation, but no adults were found to confirm their identity.

\emph{Mantel test}

The Mantel tests yielded significant results for all tests conducted
(p\textless{}0.05). The results by locality indicate the same weak
positive correlation for both software packages (Ade: r =0.1413, p
=0.0022; Vegan: r =0.1413, p =0.0032). The results for the mantel test
across the various species are not significant (Ade: r =0.0157, p
=0.4333; Vegan: r =0.0157, p =0.437).

\textbf{Discussion}

\emph{Divergence dating}

From the divergence dated phylogenies, it appears that the
genus\emph{Stasimopus} radiated in the Karoo region approximately 64.30
MYA (CI: 48.30 - 84.15), towards the beginning of the Paleocene. The
beginning of the Paleocene is marked by the Cretaceous - Paleogene
extinction event approximately 66MYA . The new niche gaps, along with
high temperatures contributed to many radiation events, which would have
aided the origin of the \emph{Stasimopus} genus .

Diversification of the genus ramps up towards the end of the Eocene and
beginning of the Oligocene (seen by the rise of clades A, B, C, G and
F). This Eocene - Oligocene boundary has been noted as `the great
divide' due to large shifts in climate (increasing temperatures in
Southern Africa), leading to changes in fauna globally . This trend of
mygalomorph diversification and geological desertification was mirrored
in Australia, which saw the radiation of the Idiopidae and
Halonoproctidae families at this time .

The genus continued to radiate in the Miocene, with the rise of the rest
of the clades, except K. The late Miocene coincides with an increase in
temperature and onset of aridification once again . This is also
mirrored in South Western Australia which experienced similar
environmental conditions and the speciation of many plants and animals .
The late Miocene and early Pliocene are noted as vitally important in
the establishment of arid zone diversity in Australia in mygalomorph
taxa such as Idiopidae (Rix et al., 2017). This same time period appears
to be important for the diversification of the arid
adapted\emph{Stasimopus} species.

\emph{Stasimopus leipoldti} (Clade K) may have arisen in the
Pleistocene, this is however a tentative conclusion due to the small
sample size of the species in this study. The Pleistocene saw Southern
Africa becoming more arid. It is also marked by alternating glacial and
interglacial cycles . These cycles are known to have had impacts on
various species ranges . There is however, evidence to suggest that
Fynbos and Succulent Karoo biomes were decoupled from these effects and
experienced climate stability . This may have led to the high levels of
diversity and endemism present in these biomes today . \emph{Stasimopus
leipoldti} is originally described from Clanwilliam (Western Cape), over
150 km away, falling into the Succulent Karoo Biome, the samples
collected for this study were from the Tankwa Karoo, which is a small,
isolated enclave of this biome nestled in the harsher Nama Karoo Biome .
This Tankwa Karoo area may thus be a Pleistocene glacial cycle refugia
for the species. A larger sample size as well as genetic sampling of the
type locality is needed to show this more definitively. The same pattern
of a Pleistocene glacial refugia was reported for the \emph{Aname} genus
in Pilbara, Western Australia, which experienced a similar geological
history to the Karoo region .

A similar relationship is seen between the type of \emph{S.
erythrognathus} (Worster, Eastern Cape) and the specimens occurring over
700 km away. This species could not be accurately dated in isolation due
to forming part of a genetic species complex. The distance between the
type locality and the specimens assessed here may indicate another
species complex, but sampling the type locality would be required to
test this.

Aridification has played a large role in shaping the evolutionary
history of \emph{Stasimopus} in the Karoo. The region is however,
experiencing rapid desertification due to climate change and habitat
degradation . This change may thus continue to drive the diversification
of the genus, or the conditions could become too harsh over too short a
time span leading to the extinction of these arid adapted species.

\emph{Demography}

The lower degree of genetic variation in the EF-1g \selectlanguage{english}region is expected
due to this being a nuclear gene and having a lower mutational rate than
the two mitochondrial genes . The high haplotype diversity across the
various species and gene regions indicates that enough time has elapsed
for diversification to occur. Majority of the species display high
haplotype diversity and low nucleotide diversity across the gene regions
(except for \emph{S. unispinosus} which does not have a high haplotype
diversity). This trend is indicative of a rapid expansion from a
relatively small population . In order to gain further insight into the
demographic history of these species for which only a few specimens were
available, more samples are required. The incongruence of the placement
of \emph{S. venterstadensis} in the haplotype network of CO1 is
potentially due to mitochondrial introgression from an ancient admixture
between the two species (Croucher et al., 2004; Horoiwa et al., 2021;
Kornilios et al., 2016). This could explain why the same pattern is not
observed in the other gene regions, but a larger sample size of \emph{S.
venterstadensis} is required to fully explore this possibility.

The EF-1g \selectlanguage{english}results for \emph{S. maraisi} indicate that the population has
undergone a recent expansion event based on the negative Tajima's D and
Fu and Li's D as well as the shape of the haplotype network This
expansion based on the dated phylogeny likely occurred in the
mid-Miocene, coinciding with the formation of the Benguela current,
leading to the Karoo experiencing a more tropical climate . This may
have given males, which are vulnerable to desiccation, more favourable
conditions to disperse further than is usually possible under arid
conditions. This could have contributed to the wide species range
of\emph{S. maraisi} seen today. The influence of the Benguela current
formation on accelerated speciation has previously been recorded in
southern Africa. In Namibia and the Northern Cape, the formation of the
Benguela current led to xeric conditions which caused local speciation
events such as the formation of the aeolian sand dune system in Namibia,
which in turn influenced the speciation of the \emph{Pachysoma} dung
beetles as well as \emph{Senecio} genus of daisies . The influence of
this current on the succulent Karoo and Fynbos biomes are also well
linked to speciation, such as on the tent tortoise, \emph{Psammobates
tentorius} as well as numerous plant species . The effect of the
Benguela on the Nama Karoo fauna, where \emph{S. maraisi} is found is
however largely unexplored, and requires further research.

\emph{Mantel test and geographic influence}

Individuals from localities which are in close geographic proximity tend
to be more related to each other than individuals which are located
further away from each other, this is supported by the results of the
Mantel test. The Mantel tests showed that there is a relationship
between geographic distance and genetic distance in our samples. There
is a weak positive correlation in both cases, meaning that with greater
geographic distance the samples are slightly less related . This is
termed isolation by distance and is a common artefact in mygalomorph
spiders due to their poor dispersal abilities and sedentary lifestyles .
This is important information to note given the possible fragmentation
of the habitat due to land use changes.

This also leads to many species being considered short range endemics
and occuring at only one or a few localities . This is true for many of
the species considered here, except for \emph{S. astutus} , \emph{S.
maraisi} , \emph{S.leipldti} and \emph{S. erythrognathus} . Most of
these species have ranges of less than 10,000 km\textsuperscript{2} This
is vital as this makes these species a conservation concern as they are
more at risk of extinction due to climate change, habitat loss as well
as habitat degredation .

The species sampled appear to experience sympatry at multiple locations.
This indicates that the species may have an unknown ecological niche
separation or are syntopic in nature . Sympatry and syntopy are common
in in disttanly related mygalomorph spiders and has been reported
several times (Leavitt et al., 2015; Satler et al., 2011; Wilson et al.,
2018). But in this case, the lineages are not as distantly related,
posing the question of whether there is a possible niche divergence and
what this may be. In the genera \emph{Aliatypus} (North America)
and\emph{Euoplos} (Australia) the niche divergence was observed in the
structure of the trapdoor lid and the habitat around the burrow (slopes
and leaf litter) (Satler et al., 2011; Wilson et al., 2017). The further
study of the species co-occuring at these localities and the habitats
may shed light on the speciation mechanisims within the genus.

\textbf{Conclusions}

\emph{Stasimopus} is an ancient group of spiders in the Karoo region.
The genus has radiated from the late Eocene and through the Miocene
resulting in 15 extant species in the region. The similarities in
biogeographic histories between the Karoo and arid region of western
Australia have seemly affected the mygalomorph fauna in similar ways.
More mygalomorph genera are needed to determine if this holds true. The
Tankwa Karoo is possibly of importance as a Pleistocene glacial cycle
refugia for the species \emph{S. leipoldti} . The area should receive
additional sampling to determine if it is a refugia for other taxa. Many
of the species are short range endemics, making them of higher
conservation concern. This is important to note as the Karoo is
undergoing further desertification due to factors such as climate
change. The genus has thrived under arid conditions, but this rapid
desertification may push the various species to their adaptational
limits.

\textbf{References}

\textbf{Data Accessibility Statement}

All genetic sequence data is available on NCBI Nucleotide Database
(MK923763-MK935687).

All specimens collected for this research have been deposited in the
National Collection of Arachnida (NCA) of the Agricultural Research
Council (ARC), Roodeplaat, South Africa.

Table 1. Comparison of the divergence dates for the various clades
(indicated on figure 2), and species under different speciation models
(tree priors) in BEAST. The mean date estimates are given in millions of
years (Ma) and the confidence intervals (based on the 95\% highest
posterior density -- HPD) are given in parentheses. The species complex
referred to is that between \emph{S. erythrognathus} and\emph{S.
karooensis} .\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Clade / lineage} & \textbf{Birth-death (Ma)} &
\textbf{Birth-death incomplete (Ma)} & \textbf{Coalescent (Ma)} &
\textbf{Yule (Ma)}\tabularnewline
\midrule
\endhead
Karoo Genus & 64.30 (48.30-84.15) & 64.31 (48.12-83.36) & 59.53
(44.62-76.51) & 94.89 (66.70-130.74)\tabularnewline
A & 37.38 (26.37-50.25) & 37.54 (26.47-50.24) & 34.11 (24.13-45.35) &
54.24 (36.75-77.67)\tabularnewline
B & 36.86 (24.92-50.78) & 36.99 (24.75-50.59) & 31.22 (22.69-40.72) &
50.97 (32.03-73.54)\tabularnewline
C & 34.31 (24.64-45.27) & 34.39 (24.89-45.12) & 33.65 (22.34-45.93) &
50.17 (34.61-71.17)\tabularnewline
D & 20.66 (13.20-29.55) & 20.69 (13.25-29.37) & 18.71 (12.07-26.65) &
29.59 (17.18-46.46)\tabularnewline
E & 18.31 (12.03-25.99) & 18.30 (11.78-25.83) & 16.57 (10.77-23.29) &
26.77 (15.40-41.66)\tabularnewline
F (\emph{S. maraisi}) & 27.30 (17.83-39.26) & 27.37 (18.11-39.32) &
24.78 (16.20-35.19) & 40.34 (23.11-63.37)\tabularnewline
G (\emph{S. hamartia}) & 32.36 (22.61-43.01) & 32.55 (22.97-43.56) &
29.55 (20.53-39.41) & 43.85 (30.09-63.41)\tabularnewline
H (\emph{S. unispinosus}) & 16.90 (10.63-24.53) & 16.94 (10.40-24.56) &
15.36 (9.40-21.96) & 24.38 (13.78-39.45)\tabularnewline
I (\emph{S. venterstadensis}) & 11.25 (6.01-17.75) & 11.28 (6.20-17.82)
& 10.21 (5.51-15.92) & 16.70 (7.66-31.49)\tabularnewline
J & 18.99 (12.44-27.42) & 19.01 (12.41-27.23) & 17.31 (11.37-24.44) &
29.19 (16.84-48.28)\tabularnewline
K (\emph{S. leipoldti}) & 0.57 (0.01-1.75) & 0.57 (0.01-1.73) & 0.51
(0.01-1.57) & 0.94 (0.24-3.21)\tabularnewline
\emph{*Species complex} & 15.76 (10.19-22.35) & 15.79 (10.18-22.34) &
14.22 (9.36-20.12) & 23.34 (14.28-50.88)\tabularnewline
\emph{S. astutus} & 11.57 (7.47-16.46) & 11.58 (7.58-16.38) & 10.49
(6.89-14.74) & 17.37 (9.92-28.48)\tabularnewline
\emph{S. dylani} & 10.29 (6.36-15.29) & 10.31 (6.37-15.29) & 9.34
(5.77-13.66) & 16.07 (8.68-26.80)\tabularnewline
\bottomrule
\end{longtable}

Table 2. Demographic parameters for the CO1 gene region for the
\emph{Stasimopus} of the Karoo region. The number of sequences (n),
nucleotide diversity (\selectlanguage{greek}π\selectlanguage{english}), haplotypes (H), gene diversity
(H\textsubscript{d}), mean number of pairwise differences (k) and
segregating sites (S) (out of 401) are given. The results of Tajima's D
and Fu \& Li's D* are also given. Significant results (p\textless{}0.05)
are indicated by an asterisk. The species complex referred to is that
between \emph{S. erythrognathus} and \emph{S. karooensis} \emph{.}
Species with less than four representatives were omitted.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\textbf{Species} & \textbf{n} & \selectlanguage{greek}\textbf{π} & \textbf{H} &
\textbf{H\textsubscript{d}} & \textbf{k} & \textbf{S} & \textbf{Tajima's
D} & \textbf{Fu \& Li's D*}\tabularnewline
\midrule
\endhead
S. astutus & 9 & 0.043 & 7 & 0.917 & 17.222 & 45 & 0.089 &
1.030\tabularnewline
\emph{S. dylani} & 7 & 0.048 & 4 & 0.857 & 19.429 & 41 & 0.472 &
0.794\tabularnewline
\emph{S. maraisi} & 36 & 0.078 & 25 & 0.973 & 23.651 & 101 & -0.682 &
0.419\tabularnewline
\emph{S. erythrognathus} & 11 & 0.068 & 6 & 0.873 & 27.382 & 55 & 0.694
& 0.896\tabularnewline
Species complex* & 12 & 0.079 & 7 & 0.894 & 22.924 & 73 & -0.090 &
0.111\tabularnewline
\emph{S. hamartia} & 10 & 0.098 & 6 & 0.889 & 37.622 & 80 & 0.776 &
0.921\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\emph{S. astutus} & \emph{S. ignis} & \emph{S.
schrieneri}\tabularnewline
\midrule
\endhead
\emph{S. dylani} & \emph{S. karooensis} & \emph{S.
theaei}\tabularnewline
\emph{S. erythrognathus} & \emph{S. leipoldti} & \emph{S.
teras}\tabularnewline
\emph{S. finni} & \emph{S. mandelai} & \emph{S.
unispinosus}\tabularnewline
\emph{S. hamartia} & \emph{S. maraisi} & \emph{S.
venterstadensis}\tabularnewline
Unidentified & &\tabularnewline
\bottomrule
\end{longtable}

\par\null

Figure 1. Map of the study area in the greater Karoo, in the
south-western part of South Africa. Markers indicate the 55 sites
where\emph{Stasimopus} specimens were found. If sites were near one
another, they are denoted as `a' and `b'. The localities are coloured
according to the species found there. Red polygons indicate areas
identified for shale gas exploration. Map created in QGIS version
3.4.8-Madeira (2019), available at:\emph{http://qgis.osgeo.org}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 2. Maximum clade credibility tree from the BEAST analysis of CO1,
16S and EF-1g \selectlanguage{english}for the \emph{Stasimopus} specimens occurring in the Karoo
under the birth-death speciation model. The grey bars indicate the
confidence intervals for the dating. Outgroups available in Table S5.
Posterior probabilities above 0.70 are given on the corresponding nodes.
Specimen numbers correspond to localities in Figure 1, and red specimens
indicate juveniles which could not be identified. Identified species are
indicated by the coloured bars. Clades are numbered for the ease of
discussion. Discussed nodes are indicated by roman numerals.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image21/image21}

\end{center}
\end{figure}

Figure 3. Map of the Karoo area sampled with haplotype networks for the
CO1 gene region. Each circle in the networks represents a
unique\emph{Stasimopus} haplotype. The black dots on each network
represents a hypothetical intermediate haplotype. Mutational steps are
indicated by lines, and more than two steps by the corresponding number.
The haplotype networks and map circles are proportional to the given
sample size.

\selectlanguage{english}
\FloatBarrier
\end{document}

