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Closely related organisms with transoceanic distributions have long been the focus of historical biogeography, prompting the question of whether long-distance dispersal, or tec-tonic-driven vicariance shaped their current distribution. Regarding the Southern Hemisphere continents, this question deals with the break-up of the Gondwanan landmass, which has also affected global wind and oceanic current patterns since the Miocene. With the advent of phylogenetic node age estimation and parametric bioinformatic advances, researchers have been able to disentangle historical evolutionary processes of taxa with greater accuracy. In this study, we used the coastal spider genus Amaurobioides to investigate the historical biogeographical and evolutionary processes that shaped the modern-day distribution of species of this exceptional genus of spiders. As the only genus of the subfamily Amaurobioidinae found on three Southern Hemisphere continents, its distribution is well-suited to study in the context of Gondwanic vicariance versus long-distance, trans-oceanic dispersal. Ancestral species of the genus Amaurobioides appear to have undergone several long-distance dispersal events followed by successful establishments and speciation, starting from the mid-Miocene through to the Pleistocene. The most recent common ancestor of all present-day Amaurobioides species is estimated to have originated in Africa after arriving from South America during the Miocene. From Africa the subsequent dispersals are likely to have taken place predominantly in an eastward direction. The long-distance dispersal events by Amaurobioides mostly involved transoceanic crossings, which we propose occurred by rafting, aided by the Antarctic Circumpolar Current and the West Wind Drift.
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Around the World in Eight Million Years:
Historical Biogeography and Evolution of the
Spray Zone Spider Amaurobioides (Araneae:
F. Sara Ceccarelli
*, Brent D. Opell
, Charles R. Haddad
, Robert J. Raven
, Eduardo
M. Soto
, Martı
´n J. Ramı
´n de Aracnologı
´a, Museo Argentino de Ciencias Naturales, Av. Angel Gallardo 470, C1405DJR,
Buenos Aires, Argentina, 2Department of Biological Sciences, 1405 Perry Street, Virginia Tech,
Blacksburg, VA 24061, United States of America, 3Dept. of Zoology & Entomology, University of the Free
State, P. O. Box 339, Bloemfontein 9300, South Africa, 4Arachnid Collection, Terrestrial Biodiversity Group,
Queensland Museum, Grey St, P. O. Box 3300, South Brisbane 4101, Queensland, Australia,
5Departamento de Ecologı
´a, Gene
´tica y Evolucio
´n, IEGEBA (CONICET-UBA), Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabello
´n II (C1428 EHA), Buenos
Aires, Argentina
Closely related organisms with transoceanic distributions have long been the focus of his-
torical biogeography, prompting the question of whether long-distance dispersal, or tec-
tonic-driven vicariance shaped their current distribution. Regarding the Southern
Hemisphere continents, this question deals with the break-up of the Gondwanan landmass,
which has also affected global wind and oceanic current patterns since the Miocene. With
the advent of phylogenetic node age estimation and parametric bioinformatic advances,
researchers have been able to disentangle historical evolutionary processes of taxa with
greater accuracy. In this study, we used the coastal spider genus Amaurobioides to investi-
gate the historical biogeographical and evolutionary processes that shaped the modern-
day distribution of species of this exceptional genus of spiders. As the only genus of the
subfamily Amaurobioidinae found on three Southern Hemisphere continents, its distribution
is well-suited to study in the context of Gondwanic vicariance versus long-distance, trans-
oceanic dispersal. Ancestral species of the genus Amaurobioides appear to have under-
gone several long-distance dispersal events followed by successful establishments and
speciation, starting from the mid-Miocene through to the Pleistocene. The most recent
common ancestor of all present-day Amaurobioides species is estimated to have originated
in Africa after arriving from South America during the Miocene. From Africa the subsequent
dispersals are likely to have taken place predominantly in an eastward direction. The long-
distance dispersal events by Amaurobioides mostly involved transoceanic crossings, which
we propose occurred by rafting, aided by the Antarctic Circumpolar Current and the West
Wind Drift.
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 1 / 20
Citation: Ceccarelli FS, Opell BD, Haddad CR,
Raven RJ, Soto EM, Ramı
´rez MJ (2016) Around
the World in Eight Million Years: Historical
Biogeography and Evolution of the Spray Zone
Spider Amaurobioides (Araneae: Anyphaenidae).
PLoS ONE 11(10): e0163740. doi:10.1371/journal.
Editor: Matjaz
ˇKuntner, Scientific Research Centre
of the Slovenian Academy of Sciences and Art,
Received: June 22, 2016
Accepted: September 13, 2016
Published: October 12, 2016
Copyright: ©2016 Ceccarelli et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All sequence data are
available form the GenBank database (accession
numbers can be found in the Supporting
Funding: This work was supported by the National
Geographic Society (http://www. research grant 7557-03
to BDO. Agencia Nacional de Promocio
´n Cientı
y Tecnolo
´gica (
research grant PICT 2011-1007 to MJR. Consejo
One of the main intrigues in biogeography has always been explaining the distribution and
evolution of closely related groups of terrestrial organisms on landmasses separatedby vast
expanses of ocean,such as oceanic islands or separatecontinents. In the case of islands that
formed from the seafloor, their current biota represents local colonisations and in some cases
radiations. Studies on the biotaof remote oceanic islands, especiallyof volcanic origin, have
been exemplary in demonstrating the ability of different organisms in colonising new areas fol-
lowing transoceanic long-distance dispersal events [13]. On the other hand, landmasses that
were separated by tectonism may harbour biota that is the result of vicariant speciation, or spe-
cies may have moved between the landmasses by over-water/aerial dispersal and evolved in
situ. An example of such landmasses are the fragments of the supercontinent Gondwana,
which began breaking up during the early Jurassic, approximately 190 million years ago [4].
The separation of the last part of the Gondwanan landmass, namely Australia-Antarctica-
South America during the Miocene, gave rise to the Antarctic Circumpolar Current in the
ocean and the West Wind Drift in the atmosphere [5]. The Antarctic Circumpolar Current
and West Wind Drift have been key factors affecting the directionality of long-distance dis-
persal events in the Southern Hemisphere, particularly for organisms that disperse by rafting
(or oceanic drift) and wind, creating what has been termed dispersal asymmetry” for a pre-
dominantly eastward dispersal pattern [69].
Spiders, amongst thearachnids, are generally consideredof high vagility due to their dis-
persal capacity by the phenomenon termed ballooning, by which juveniles can be carried long
distances through the air by a strand of silk released from their spinnerets (see [10]). Not only
can many spiders undergo long-distance dispersal by this mechanism, but some species have
also been shown to withstand contact with water, and even take-off from the surface of salty,
turbulent water [11], a crucial feature for surviving transoceanic dispersal. Studies of spiders on
different oceanic islands have shown that they are among the first and most successful coloni-
sers, with the lineages at times arising from multiple dispersal events [1215] (Soto pers. obs.).
However, for some spiders and other arachnids, which lack thepropensity for ballooning, an
alternative explanation must be provided for groups with a transoceanic (or volcanic island)
distribution. Certain globally distributed groups of arachnids with poor dispersal capacities
(e.g. opilionids, [16], or palpimanoid spiders, [17]) have diverged through tectonic related
vicariant cladogenesis, yet in other cases the divergence times do not coincide with landmass
fragmentations [18]. As the alternative to aerial dispersal, rafting must also be considered for
such spiders. Spiders have been observed on vegetation (macrophyte) rafts in the Amazon [19]
and rafting as a means of transoceanic dispersal has been suggested for mygalomorphs [20],
trapdoor spiders [21], salticids [22], the genus Dysdera [23], the desid Desis marina [24] and
the anyphaenid genus Amaurobioides O. Pickard-Cambridge, 1883 [25,26].
Among the spiders, several species of the family Anyphaenidae are known to disperse
through ballooning in the Northern Hemisphere [27,28], but not Amaurobioides (Ramírez,
pers. obs.; see below). The anyphaenid genus Amaurobioides is exceptional for its ecology and
distribution. According to the detailed study by Lamoral [29], Amaurobioides africana Hewitt,
1917 seal their retreats to endure daily periods of immersion as the tides rise. With low tide
they open the entrance of their silken cells, and at night prey mostly on isopod and amphipod
crustaceans. The individualsprey from the cell entrance, not walking away from their retreat as
other spiders do. The species of Amaurobioides from Chile, Australia and New Zealand posi-
tion their retreats in the spray- rather than the intertidal zone, thus are probably immersed less
often than the population studied by Lamoral, but still seal their retreats in a similar way [30
33]. To date, 12 species have been described from rocky shores in South Africa (one species),
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 2 / 20
Nacional de Investigaciones Cientı
´ficas y
´gicas (
BecaPosdoc13 to FSC.
Competing Interests: The authors have declared
that no competing interests exist.
Australia (two species), New Zealand (eight species) and Chile (one species) [29,30]. However,
the number of species from New Zealand is probably over-estimated, since there appear to be
at most three genetic lineages [32,33].
While reviewing the spiders of the subantarctic islands of New Zealand, Forster [24]
thought that Amaurobioides was composed of a single species widespread in South Africa, Aus-
tralia and New Zealand, that dispersed easily across sea expanses. He later revised the genus,
finding morphological differences to distinguish no fewer than 10 species, thus suggesting that
their dispersal ability has been overestimated [30]. Later on, Forster & Forster [34] attributed
their Gondwanic distribution to ancient vicariance, while Hewitt [35] had mentioned the possi-
bility of the species’ passive dispersalon floating seaweed. While thedistribution of this genus
is Gondwanan in the sense that they are found on continental shelves once forming part of
Gondwana, the question that arises is whether this distributional pattern can be attributed to
ancient vicariance from the separation of the landmasses or whether more recent, long-dis-
tance dispersal events were involved in the species’ distributions. On a smaller scale, phylogeo-
graphic studies of Amaurobioides from Australia and New Zealand have revealed their
propensity for dispersal between the two landmasses [26], mainly from Australia towards New
Here, we present a parametric biogeographical study of Amaurobioides species from Africa,
Australia, New Zealand and South America based on molecular phylogenetic species tree anal-
yses performed using two mitochondrial (cytochrome oxidase c subunit 1 and 16S rDNA) and
two nuclear (Histone 3-a and 28S r-DNA) gene fragments, to shed light on the genus’ geo-
graphic range evolution and compare it to other global biogeographical and evolutionary
Materials and Methods
Taxon Sampling
A total of 45 Amaurobioides individuals were used for molecular work in this study, of which
34 were assigned to the species A.africana (20 individuals from South Africa), A.chilensis (3
individuals from Chile), A.isolata Hirst, 1993 (1 individual from South Australia), A.litoralis
Hickman, 1949 (1 individual from Tasmania, Australia), A.maritima (2 individuals from New
Zealand), A.pallida Forster, 1970 (3 individuals from New Zealand) and A.pleta Forster, 1970
(4 individuals from New Zealand). The remaining 11 individuals belonged to undescribed spe-
cies from South Africa (7 individuals) and Flinders Island, Tasmania (4 individuals). This sam-
pling covers almost the entire known distributional range of the genus, with the exception of
the subantarctic Auckland and Campbell islands of New Zealand, where A.piscator Hogg,
1909 is found. Collecting details of all Amaurobioides individuals used for molecular work can
be found in Table A in S1 File. Collecting and export permits for New Zealand were issued by
New Zealand’s Department of Conservation. For Amaurobioides species collected in Australia,
the South Australian Department of Environment and Natural Resources and the Tasmanian
Department of Primary Industries, Parks, Water, and Environment issued collecting permits,
and the Australian Wildlife Trade Assessments provided the export permit. Further specimens
from Tasmania were collected on Bush Blitz ( expeditions with National
Park permits provided by Tasmanian National Parks and Wildlife Service. For the South Afri-
can specimens, collecting permits were obtained from CapeNature (Western Cape Province)
and the Eastern Cape Department of Economic Development and Environmental Affairs
(Eastern Cape Province). Amaurobioides chilensis was collected in unprotected public areas in
Chile, where permits are not needed. None of the field studies involved endangered or pro-
tected species.
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 3 / 20
As sister- and outgroup taxa for Amaurobioides, we selected 60 anyphaenid taxa based on
previous molecular phylogenetic studies (Soto pers. obs.) [36]. We chose one specimen per spe-
cies of the following anyphaenid genera belonging to the tribe Amaurobioidini of the subfamily
Amaurobioidinae: Acanthoceto (5 species), Axyracrus (1 species, monotypic), Aysenia (3 spe-
cies), Aysenoides (4 species),Coptoprepes (3 species),Ferrieria (1 species, monotypic), Gamakia
(1 species, monotypic), Negayan (3 species), and Selknamia (1 species, monotypic). From the
subfamily Amaurobioidinae, tribe Gayennini, we chose one specimen per genus for the genera
Arachosia,Araiya,Gayenna,Gayennoides,Monapia,Oxysoma,Phidyle,Tasata and Tomo-
pisthes, two species belonging to Sanogasta and one specimen per species from the genus Phi-
lisca (15 species), to ensure that the age of the mrca of Philisca species endemic to Juan
Fernandez Island was less than that of the island (Soto pers.obs.). The genus Josa, a member of
Amaurobioidinae not assigned to a tribe, was represented by J.calilegua,J.riveti and an unde-
scribed species (Josa sp.). Additionally, 11 anyphaenid species belonging to nine genera from
the subfamily Anyphaeninae were used as outgroup taxa and for fossil node age constraints
(see below): Anyphaena,Anyphaenoides,Aysha,Buckupiella,Hatitia,Hibana,Jessica,Otoniela
and Xiruana. A specimen belonging to the clubionid genus Elaver was used for rooting the
trees, following the molecular results of the Spider Tree of Life project (Ramírez et al., in prep),
which show that the Chilean genus Malenella, formerly considered a member of Anyphaeni-
dae, belongs elsewhere. For molecular phylogenetic analyses, a combination of DNA sequences
from previous studies (Soto pers. obs.) [36] and sequences generated de novo was used (for
details of specimens and sequences used please see Table A in S1 File).
DNA extraction, PCR and Sequencing
For the sequences generated de novo for this study, DNA was extracted from leg muscle tissue
using the Qiagen DNeasy Blood and Tissue Kit, following the manufacturers protocol and
digesting the tissue at 56°C over-night with Proteinase K. Polymerase Chain Reaction (PCR)
mixes contained 1.5μl x10 PCR Buffer (Thermo Scientific), 10 μmoles MgCl
, 0.25 μmoles of
each dNTP, 0.4 μmoles of each primer, 0.1 μl Taq Polymerase (Thermo Scientific), 0.5 μl BSA,
1–2 μl genomic DNA and ddH
O to bring the final volume to 15 μl. The primers used for
amplification can be found in Table B in S1 File. Thermal cycling profiles included an initial
denaturing stepat 95°C for 3 minutes, followed by 15 cycles of 30 secondsat 95°C, 30 seconds
at the annealing temperature (51°C for nuclear and 45°C for mitochondrial gene fragments)
and 45 minutes at 72°C;an additional 20 cycles were then runwith the annealing temperature
lowered by 3°C. A final extension step of 10 minutes at 72°C was then set. PCR products were
purified using ExosAP (Thermo Scientific) following the manufacturer’s indications and sent
for sequencing to Macrogen Inc., Korea. Sequences were edited based on the chromatograms
in Sequencher v. 4.1.4 (GeneCodes corp.), where each protein-coding gene fragment was
checked for stop-codons (indicating possible pseudogenes).
Phylogenetic Inference and Divergence Dating
The edited sequences and the sequences obtained from previous studies were aligned to pro-
duce one data matrix per gene fragment. Matrices for divergence dating included all of the out-
groups mentioned previously to allow for node age calibrations, whereas the remaining
analyses were carried out with Aysenia elongata Tullgren, 1902 and Coptoprepes campanensis
Ramírez, 2003 as the only outgoups. Alignment for the Histone 3-a (henceforth H3a) gene
fragment was straightforwardand was carried out in the online version of MAFFT v. 7 [37]
using the “Auto strategy and a gap opening penalty of 1.53. The cytochrome oxidase c subunit
1 (henceforth COI) gene fragment contained indels and was therefore aligned based on its
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 4 / 20
translation to protein in TranslatorX [38] while for the 16S rDNA (henceforth 16S) and 28S r-
DNA (henceforth 28S) gene fragments the online version of T-coffee was used [39,40] using
the default settings, that take into account secondary structure of ribosomal DNA during align-
ment. Poorly aligned positions for the 16S matrix were removed using the Gblocksserver [41]
allowing for gap positions and less strict f lanking positions in the final alignment. Furthermore,
recombination in nuclear genes has been shown to interfere with reliable topologicalinferences
[42], so we tested interspecific recombination for Amaurobioides using the Maximum Chi-
square method in RecombiTEST [43], using only non-recombining segments for both nuclear
markers (H3a and 28S).
Nucleotide compositional homogeneity within each data matrix was tested using the chi-
squared metric provided in the program TreePuzzle [44], to evaluate the molecular marker’s
utility for phylogenetic reconstruction at the taxonomic level intended (family level for the
complete-outgroup dataset and genus level for the Amaurobioides dataset with two outgroup
taxa). Upon confirmation that all sequences in the alignments had passed the nucleotide com-
positional homogeneity test, data partitioning strategies and nucleotide substitution models
were selected using PartitionFinder v. 1.1.1. [45] (see Table C in S1 File for details). Further-
more, the net evolutionary distances between Amaurobioides species were estimated for each
fragment in MEGA7 [46].
A separate phylogenetic tree was obtained for each gene fragment to evaluate gene tree dis-
cordances, after whichthe matrices for the gene fragments were analysed together to obtain a
concatenated phylogeny and to estimate node ages, using the taxa for which at least 3 out of
the 4 markers were available. Phylogenetic trees were obtainedby Bayesian Inference (BI) in
MrBayes v. 3.2.3 [47] applying a Markov Chain Monte Carlo (MCMC) simulation of 20 mil-
lion generations, sampling a tree every 2,000
generation for four chains in two independent
runs. The data was partitioned and separate nucleotide substitution models were set as priors
for the partitions based on the scheme and models selected by PartitionFinder and parameters
were unlinked across partitions.Once the stationarity and correct mixing of the MCMC runs
was confirmed, consensus trees were then built for each analysis using the “sumt command
discarding the first 25% as burn-in. Nodal support was evaluated through posterior probability
Divergence time estimates with node calibrations were carried out to obtain rates for the
mitochondrial gene fragment to be used in the species tree analysis (see below), using the
concatenated dataset with all outgroups, by BI with MCMC simulations in the program
BEAST v. 1.8.2 [48], partitioning the data by marker, unlinking the substitution and clock
model priors for each partition and setting the most appropriate substitution model (as deter-
mined by PartitionFinder) to each partition. For the tree prior a Birth-Death process was cho-
sen (appropriate for the super-specific nature of our data) and an uncorrelated lognormal
relaxed clock prior was set for each partition and the rates estimated based on a lognormal dis-
tribution around the mean (0), with an initial value and standard deviation of 1, providing a
permissive range for the program to auto-optimize towards the true posterior values.
Information from two fossil anyphaenids was used to apply node age priors to the analysis.
A fossil specimen in Baltic amber—estimated to be between 33.9 and 37.2 Myr old and postu-
lated to belong to the genus Anyphaena—was used to set a uniform prior with a minimum age
of 33.9 Myr [49]. We used this minimum age as a constraint for the mrca of anyphaenines in
our study, since the assignment of this immature specimen to Anyphaena is not certain [50],
and considering that our sampling of extant species belonging to this genus is not extensive.
Information from a second fossil specimen belonging to the genus Anyphaenoides from
Dominican amber [50] was used to set a uniform prior witha minimum age of 13.65 Myr to
the mrca of Anyphaenoides and its sister group taxa (given the fact that only one specimen of
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 5 / 20
Anyphaenoides was available). Two independent runs of 80 million generations each (sampling
every 8,000
) were carried out to confirm that there was convergence. The outcomes of the
two runs were also validated in Tracer v. 1.5 [51] to ensure that the effective sample sizes of the
parameters were greater than 200. The tree files were then combined using LogCombiner1.8.2
(part of the BEAST package) and the maximum clade credibility (mcc) tree with mean node
heights selected in TreeAnnotator 1.8.2, setting the burn-in at 10%. Nodal support was assessed
based on posterior probability, and the precision of the node age estimates were evaluated by
comparing the ages to known events (the appearance of Juan Fernandez for endemic Philisca
species, see [52] and Soto pers. obs.) and the age-calibrated rates to values found in the litera-
ture for the same molecular markers.
Species Tree Inference
As gene-tree-species-treediscordance can present a major problem for obtaining a topology
that accurately reflects the species’ evolutionary histories [53,54], a multispecies coalescence
approach was applied for Amaurobioides, using two outgroup taxa (Aysenia elongata and Cop-
toprepes campanensis). Since mitochondrial genes are presumed to be linked, we combined the
COI and 16S fragments for the analyses. Species assignation was based on a combination of pre-
vious results and taxonomic status, so even though A.pleta and A.pallida were not always recip-
rocally monophyletic, based on the (albeit low) genetic divergence of COI (see results) and in
the absence of a taxonomic revision to date, we decided to treat them as separate species.
The coalescence-based species tree analysis was carried out in BEAST v. 1.8.2 using the
BEAST (starBEAST [55]) implementation on the mitochondrial and two nuclear fragments,
applying an uncorrelated lognormal relaxed clock prior to each fragment (mitochondrial, H3a
and 28S), and setting the mean of the clock rate prior (ucld.mean, with a normal distribution)
for the mitochondrial fragment at 0.01551 based on the geometric mean of the rates estimated
by the concatenated BEAST analysis and setting the standard deviation of the normal prior at
0.3 to allow for leniency in the node-age estimation. Additionally, the node representing the
mrca of Amaurobioides +Aysenia elongata was constrained and a normaldistribution was cho-
sen for the age prior, with a mean±SD of 10.9773±1.35 to obtain the same 95% confidence
interval values as for the dated concatenated analysis (see Results). Nucleotide substitution
models were set for each of the three partitions according to the PartitionFinder results and the
tree prior was set to a Birth-Death process. Four independent MCMC chains of 100 million
generations (sampling every 2,000
) were run, verifying the runs and selecting the maximum
clade credibility tree as for the divergence dating analysis.
Due to the fact that some species in this study were represented by a single individual, and
species coalescence analyses rely on several individuals per species to obtain estimates of
parameters such as population size, the BEAST analysis was re-run with the same settings and
parameters as above, but removing the taxa with one individual per species (A.isolata,A.litor-
alis and A. sp. Flinders). This re-run served the purpose of verifyingthe extent to which these
single individuals affected node age estimates. All BI runs were carried out via the CIPRES Sci-
ence Gateway v. 3.3 [56].
Tests of Monophyly
The marginal likelihoods of different hypothetical topologies were compared using steppingstone
sampling [57] in MrBayes, to determine the most likely phylogenetic position of A.chilensis from
South America with regards to the species from Australasia (Australia and New Zealand). Five
runs with alternative constrained topologies were executed (see Fig 1), where the topologies were
constrained to obtain all possible hypothetical positions of A.chilensis with regards to the species
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 6 / 20
from Australasia, maintaining only the well-supported sister-group relations intact. Stepping-
stone sampling wasset to run for 50 steps with 10 milliongenerations. For marginal likelihood
comparison, Bayes Factors were used to assess the support of the difference in log likelihoods [58]
between the run with the highest marginal likelihood value and the remaining runs.
The same alternative topologies were compared using parsimony with TNT [59], with a
search strategy of 100 random addition sequences, each followed by TBR (commands "mult
Fig 1. Alternative topologies for testing the phylogenetic placement of Amaurobioides chilensis.Schematic diagrams of five
topological scenarios tested by marginal likelihood comparison obtained through steppingstone sampling in MrBayes for Amaurobioides,
with the monophyly constrained for alternative nodes. The letter “M” indicates the nodes for which monophyly was constrained, with
parsimony bootstrap percentages below. The tree terminals contain the continent in uppercase letters (with AU for Australia and NZ for
New Zealand in brackets) and the species names in italics underneath. Sister species relations which were supported in the original
analyses are kept as a single terminal in the schematic diagrams.
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 7 / 20
100;"). Tree lengths were obtained all the constrained analyses as above. The frequencies of
constrained groups were also calculated in 1000 bootstrap replicates of the unconstrained data-
set using the command “majority [x]” of TNT.
Ancestral Area and Event Estimation
Ancestral ranges and speciation events for Amaurobioides were estimated in R version 3.2.2
[60] using the “BioGeoBEARS” v. 0.2.1 package [61,62], which integrates and compares the
Dispersal-Extinction-Cladogenesis (“DEC” [63]); DIVALIKE, modified from the DIVA pro-
gram [64]; and BAYAREALIKE, modified from the BayArea program [65] algorithms, adding
an extra parameter (“j”) to each method, which models “jump dispersal” or founder event spe-
ciation [66]. The following seven areas were used in the analyses: South Africa (AF), South
America (AM), South Australia (AU), Tasmania (AT), southern South Island of New Zealand
(NS), North Island + northern South Island of New Zealand (NN) and Antarctica (AN), to
consider the possibility of Antarctica as one of the ancestral areas. The areas NS and NN were
chosen based on prior information of the distributional ranges of Amaurobioides in New Zea-
land [32,33].
Since the relative positions of the continents have not changed much during the time-frame
of the analyses (see Results), a time-stratified analysis was not implemented. An area adjacency
matrix was provided, setting areas as “adjacent” even when separated by the sea, but not if
another area from this study was found in between (i.e. any one area could only have a maximum
of three adjacent areas). Additionally, a distance matrix was built based on the distances between
the furthest known points of distribution for Amaurobioides in each area and dividing the values
by 10,000 (so that the greatest distance—10,000 km—would equate to 1 and any smaller distance
would be expressed as a fraction between 0 and 1). Four different runs were executed to compare
the log likelihood of different dispersal scenariosbased on modifications made to the dispersal
multiplier matrix. The firstdispersal multiplier matrix was set as “unconstrained”, giving a prob-
ability of 1 for dispersal to occur between all areas. The second dispersal multiplier matrix was
based purely on distances, subtracting the distance fraction from 1, to leave the greater distances
with lower dispersal probabilities and vice-versa. The following two dispersal multiplier matrices
were set to allow only dispersal from West to East (“Eastward dispersal”) or from East to West
(“Westward dispersal”). All input matrices for BioGeoBEARS used in this study can be found as
Matrices A-D in S1 File. Likelihoods between the scenarios and runs were compared via Bayes
Factors to select the method and scenario which best explains the data.
Although the species tree was considered to provide the most reliable topology based on the
aforementioned computational advantages, a second biogeographical analysis was carried out,
considering the possibility of an alternative topology as inferred by the concatenated data in
BEAST (and corroboratedby the tests of alternative topologies), particularlyregarding the sister-
group relations between species from Australasia and the South American A.chilensis. The dated
tree was edited in Mesquite v. 3.04 [67] to use as an input for BioGeoBEARS. The settings and
priors for the ancestral range estimation analyses were set as above, except for the areas, which
were reduced to four by combining the Australian and New Zealand areas into one, re-naming it
Australasia. This reduction was applied because in this case the analyses were only run to corrob-
orate the ancestralrange and event estimations betweenAustralasia and South America.
Phylogenetics, Species Tree Inferences and Divergence Dating
The aligned DNA data matrices used for phylogenetic inferences contained 657, 357, 229 and
157 characters for the COI, 16S, H3a, and 28S gene fragments,respectively, after removal of
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 8 / 20
hypervariable regions and recombining segments. Of these characters, the number of variable
and parsimony-informative sites respectively for matrices including outgroup taxa were 292
and 245 for COI, 183 and 142 for 16S, 91 and 71 for H3a and 57 and 38 for 28S. The number of
variable and parsimony-informative sites respectively for Amaurobioides sequences only were
139 and 118 for COI, 49 and 43 for 16S, 26 and 15 for H3a and 10 and 7 for 28S, reflectingthe
low evolutionary divergence based on the nuclear fragments between Amaurobioides species,
especially those from Australia, New Zealand and South America (see Table D in S1 File).
Based on the gene trees, it is also clear that the nuclear markers lack sufficient information nec-
essary to fully resolve interspecific relationships of Amaurobioides, while the mitochondrial
markers contain more information, sufficient for resolving the deeper nodes within the genus,
including when the data is concatenated (Fig 2 and Figures A to E in S1 File).
Fig 2. Phylogenetic tree of Amaurobioides inferred by MrBayes for the concatenated data. Tree inferred using COI, 16S, H3a and
28S data, obtained by 50% consensus of 10,000 trees. Bayesian posterior probabilities (PP) >= 0.9 are represented as circles at the
nodes (black: 1<= PP<0.95; white: 0.95<= PP<0.9) and bootstrap support values from 1,000 replicates on the tree obtained by parsimony
analysis, are shown to the left of each node. Missing values indicate the clade was not recovered.
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 9 / 20
Based on the species tree coalescence analysis (Fig 3), the monophyly of Amaurobioides is
supported, with a divergence of the most recent common ancestor (mrca) estimated between
4.95 and 9.94 Ma, 95% Highest Posterior Densities (HPD). The two species from South Africa
appear to have diverged between1.30 and 5.06 Ma, 95% HPD, and were found to be sister to
the remaining species of the genus. The monophyly of the species from Australia, New Zealand
and South America is also supported, with the mrca diverging approximately 4.38 Ma (95%
HPD: 2.30–6.22 Ma), yet species relations within this clade are generally poorly supported. The
only sister group relations supported within this group are A.pallida +A.pleta (from New Zea-
land) and A.litoralis +Amaurobioides sp. from Flinders Island (both from Tasmania), esti-
mated to have diverged 0.96 and 0.43 Ma, respectively. Removing the species with a single
representative from the analyses had no major effect on node age estimates, as can be seen
from the 95% HPD for both trees (see Figure F in S1 File).
The marginal likelihoods (in log units) for the tests of alternative topologieswithin the Aus-
tralasia+South America clade can be found in Table 1. Taking into account that the support for
the hypothesis with the higher log likelihoodvalue is considered positive if twice the difference
in log likelihoods(i.e. the Bayes Factor) is greaterthan 2 [57], the most likely scenario with pos-
itive to strong support in this case is that the species from South America falls within the clades
from Australasia (as for the concatenated analyses, Fig 2 and Figure E in S1 File). The least
likely scenario would be A.chilensis from South America as sister to a clade from Australasia
(scenario 1). Similarly, in the parsimony analysis the alternative resolution with A.chilensis as
sister to the Australasian species is the least parsimonious (see Table 1), and never appears in
Fig 3. Time-calibrated species tree and photographs. Time-calibrated species tree of Amaurobioides using *BEAST shown on the left.
Blue node bars represent 95% Highest Posterior Density intervals for node ages. Bayesian posterior probability values are shown at
nodes. Codes in brackets next to terminal taxa names correspond to their areas of distribution (AM = South America; AF = Africa;
AU = South Australia; AT = Tasmania; NN = North Island and northern part of South Island of New Zealand; NS = central and southern part
of South Island of New Zealand). Photos to the right of the tree are of Amaurobioides maritima female (top left; photo M.J. Ramı
´rez), two A.
maritima retreats from Jackson Bay, South Island, New Zealand (top right; photo B.D. Opell) and typical habitat of A.maritima, Waikawa,
South Island, New Zealand (bottom; photo M.J. Ramı
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 10 / 20
the bootstrap pseudoreplicates (Fig 1), whereas scenario 2 received the lowest tree-length value
and of all the scenarios is therefore considered the most parsimonious.
The node age estimates obtained from the concatenated matrices were generally older,
which has been noted and justified previously [68]. The divergence of the mrca of Amauro-
bioides was estimatedat 10.08 million years ago (Ma; 7.52–12.58, 95% HPD; Figure E in S1
File). Based on these node age estimates, the species from South Africa appear to have diverged
around 5.79 Ma, while the sister group containing the species from Australia, New Zealand
and South America began diversifying around 6.44 Ma. The split between A.pleta and A.pal-
lida was estimated at around 1.03 Ma, and between A.litoralis and Amaurobioides sp. from
Flinders Island around 1.28 Ma. Based on this tree, the relations between the Australian, New
Zealand and South American taxa are not fully resolved either. The mean rates obtained for
the four markers used in this study are comparable to molecular rates obtained for the same
markers from different studies (see Table E in S1 File). Furthermore, the divergence of the Phi-
lisca species endemic to Juan Fernandez Island was estimated at 1.59 Ma (1.03–2.18, 95%
HPD), therefore younger than the island itself (consistent with the volcanic origin of the island
4 Ma), as expected (Soto pers. obs.). We therefore considerthe node age estimates for Amauro-
bioides reliable for the purpose of this study.
Ancestral Area and Event Estimation
The BioGeoBEARS run, using the species tree, with the highest log likelihood values for most
models was the “Eastward dispersal” run, with the dispersal multiplier matrix set to favour dis-
persal from the West to the East and within each run. Additionally, the DIVALIKE+J model
was always favoured over the others (see Table F in S1 File), although the difference in log like-
lihoods with the BAYAREALIKE+J run was minimal. Based on the DIVALIKE+J outcome for
the “Eastward dispersal” run, the ancestral area of Amaurobioides was Africa. Since the remain-
ing genera of Anyphaeninae (outgroups to Amaurobioides in this study) are distributed in
South America, a dispersal event from South America to Africa during the Miocene is likely to
have established the ancestral populations of Amaurobioides (see Fig 4). Founder-effect type
events following long-distance dispersal from Africa to Australia during the late Miocene most
likely established the ancestral area of the Australia + New Zealand + South Americaclade.
Since the phylogenetic relationships within this group were not resolved, most biogeographic
event estimations for this clade cannot be commented on with certainty. However, even based
on the most likely BioGeoBEARS run (Table G in S1 File) using the alternative topology from
the concatenated data, ancestral populations of A.chilensis were established in South America
after a long-distance dispersal event from Australasia (Figure G in S1 File). Furthermore, the
re-colonisation of South America from Australasia is highly likely based on several lines of evi-
dence discussed below.
Table 1. Marginal likelihoods in log-units (lnL), Bayes Factors (BF) and parsimony tree lengths (PL)
for the alternative topological scenarios tested for Amaurobioides as depicted in Fig 1, obtained by
steppingstone sampling in MrBayes and topological comparisons in TNT. Asterisks indicate the sce-
nario which received the highest log likelihood value.
Scenario lnL BF PL
Scenario 1 -3065.65 13.62 426
Scenario 2 -3061.52 5.36 419
Scenario 3 -3062.18 6.68 424
Scenario 4 -3058.84 *** 424
Scenario 5 -3065.21 12.74 421
Amaurobioides Long-Distance Eastward Dispersal
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Fig 4. Biogeographical areas and events estimations. Biogeographical areas and events estimations obtained from the West Wind
Drift-constrained run with the DIVALIKE+J algorithm. Coloured symbols at the tips of the species tree represent the current sampling
localities of the specimens used in this study, shown also on the bottom map using the same colours and shapes. Additionally, codes in
brackets by the tips of the species tree correspond to the areas of distribution (AM = South America; AF = Africa; AU = South Australia;
AT = Tasmania; NN = North Island and northern part of South Island of New Zealand; NS = central and southern part of South Island of
New Zealand). Pie charts at the nodes of the species tree represent the relative probabilities of the ancestral areas, while pie charts at the
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 12 / 20
Phylogenetic Inferences
In this study the relationships between Amaurobioides species were only fully resolved at a
deeper level and in some derived sister group relationships. The poor resolution within the
Australian and New Zealand clade is probably due to the low variability, i.e. phylogenetic sig-
nal, of the nuclear markers. A recent phylogeographic study [26] disentangles the relationships
between Australian and New Zealand speciesusing denser sampling and different molecular
markers. Adding more taxa and especially more (rapidly-evolving) molecular markers may
provide a clearer picture of the evolutionary processes that have shaped the currentdiversity in
said areas. The coastal areas between South Australia (where the A.isolata specimen was col-
lected) and south-eastern Australia, just north of Tasmania and Flinders Island, appear prom-
ising for more Amaurobioides diversity still to be discovered. Perhaps this missing information
may explain some of the low support for internal branches of the phylogenies, which would be
better resolved with a broader sampling from Australia.
Regarding the Amaurobioides species from New Zealand, even though there are eight
described species to date, their validity is questionable [26,32,33]. With this in mind, the spe-
cies used in this study represent the genetic diversity found on the North and SouthIslands of
New Zealand. The only valuable addition would therefore be A.piscator from New Zealand’s
Sub-Antarctic Auckland and Campbell islands [25,30,33]. Future systematic contributions
will include a revision of New Zealands’ Amaurobioides as well as formal descriptions of the
undescribed species used in this study.
Historical Biogeography
The ancestral area of the genus Amaurobioides was estimated to be southern Africa, upon the
establishment of (an) ancestral population(s) during the Miocene from South America, consis-
tent with the beginning of the Arctic Circumpolar Current [5]. Since the Gondwanan conti-
nents had separated by the Miocene,the ancestral population that gave rise to the mrca of
Amaurobioides is likely to have arrived to Africa from the west by long-distance dispersal. The
relatively deep divergence between the species found in South Africa are further evidence sup-
porting the long time span of this genus’ presence in the continent, and therefore its plausibility
as the ancestral area. South Africa, in particular the Cape area, is well-known for its extremely
high level of species richness and endemism of plants [69], which have undergone numerous
long-distance dispersal events to and from South Africa [9]. Apart from plants, the African
continent is the ancestral area, and thesource of long-distance dispersalevents, for other
organisms, including nymphalid butterflies [70], thrushes [71], platyrrhine primates [72], and
with the findings of the present study, the genus Amaurobioides as well.
Further eastward long-distance dispersal events of Amaurobioides from Africa to Austral-
asia (Australia and New Zealand) around the Mio-Plioceneboundary and the mid-Pliocene
were estimated in this study, establishing present-day species in South Australia, Tasmania,
North Island and South Island of New Zealand. Nevertheless, the aforementioned sampling
gap from Australia’s south-eastern coast and the absence of A.piscator may not only have
corners represent the relative probabilities of founder-effect dispersal to new areas. Smaller maps to the right of the species tree represent
historical events during given epochs, with arrows representing estimated long-distance dispersal events. Bottom map contains the
sampling localities of the specimens used in this study, colour and shape coded as in the terminal branches of the tree. Arrows represent
dispersal events inferred in this study (solid line: ancestral Amaurobioides species; dashed line: mrca of Amaurobioides). The grey
box around Australia and New Zealand indicates that the exact events within those areas remain unresolved. The red oval marked A+C
shows the position of Auckland and Campbell Islands.
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 13 / 20
weakened nodal support, but also obscured other possible events regarding Amaurobioides in
said areas during the Pliocene. Alternatively, the low nodal support may not be a cause for
diluted biogeographical signal, but an effect of it, if repeated dispersal between Australia and
New Zealand gave rise to prolonged periods of genetic introgression and hybridisation, as may
be expected between adjacent landmasses [7]. As populations then established and speciated, it
is likely that secondary colonisation became more complicated [6,73]. The divergence of the
mrca from Australia and New Zealand, estimated at around 4.36 Ma is similar to previously
suggested ranges (4.0–4.6 Ma [32]; 4.497 Ma: mrca of Australian + NZ species [26]). While it is
still unclear whether the species from New Zealand are monophyletic (this study’s species tree,
[32,33] or not [26], their biogeographical history remains to be thoroughly tested.
The postulated Plio-Pleistocene boundary colonisation of South America from New Zea-
land by Amaurobioides found in this study in a sense closes the circle of long-distance dispersal
followed by colonisation events and speciation of this genus. Although the sister group relation
of A.chilensis and A.maritima was not supported by molecular evidence in this study, the
topological tests allow us to rule out a monophyletic group from Australasia and therefore lend
support to an Australasian origin for the South American species. In addition, morphological
evidence lends support to the sister group relation of A.chilensis and A.maritima, as the
females of the two species are virtually indistinguishable [29]. Furthermore, since Amauro-
bioides is only distributed along the west coast of South America, its colonisation from the east
is more likely than colonisation, range expansion and local extinction from the eastern or
southern parts of South America. Specimens of A.chilensis have been found in localities along
the Chilean coast at least 1200 km apart [31]. A more in-depth phylogeographic study of A.chi-
lensis would therefore be necessary to trace its colonisation and expansion history, as well as
estimating the species’ genetic diversity since the early Pleistocene.
Other possible explanations for Amaurobioides species’ dispersal routes between continents
are less plausible and would require further evidence. For example, the role of Antarctica in the
genus’ biogeographicalhistory is difficult to conceive, due to its coasts beingcurrently unin-
habitable for Amaurobioides. However, it may be that during warmer periods of the earth’s his-
tory (late Miocene/early Pliocene), Antarctica may also have harboured ancestral populations,
presenting an additional geographic range for dispersal to- and from. Including A.piscator in
the analyses may shed light on more southern events, in that we could infer the direction from
which they reached Auckland and Campbell Islands, although considering the geographic
position of these islands, they are approximately five times closer to New Zealand than to Ant-
arctica and therefore the spiders are more likely to have arrived from New Zealand. In any
case, only fossil evidence from Antarctica itself would unambiguously lend support to the pres-
ence of past populations or species on the continent, since even the likelihood-based bio-
geographical analyses of this study never estimated Antarctica as an ancestral area.
Despite a few uncertainties in the phylogenetic results, the overwhelming patterns found in
this study support repeated eastward long-distance dispersal events by ancestral Amauro-
bioides populations acting as founder events in distant coastlines of other continents. This east-
ward trend fits inwith predictions based on theAntarctic Circumpolar Current and the West
Wind Drift. The West Wind Drift has been used to explain the distribution of wind-dispersed
organisms in the Southern Hemisphere, particularly plants [8,9]. Unlike many widely distrib-
uted genera of small spiders, well-known to disperse by aerial ballooning [74], we find it
unlikely that Amaurobioides disperse by that mechanism. It has been shown that species that
are specialists of fragmented habitats have a low propensity for ballooning [75], and Amauro-
bioides use a narrow, highly fragmented niche, where almost any wind would take them away
to the water, land, or unsuitable sandy beaches. Other circumstantial evidence supports this
reasoning. Amaurobioides specimens were collected by probing them out of their cells with a
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 14 / 20
metal wire; as the spiders are forced out they gasp firmly on the rock surface, run or hide, but
never jump (MJR, BDO, CRH, pers, obs.).This behavior is in contrast with theusual escape
strategy of most entelegyne spiders and anyphaenids in general, of jumping away or dropping
while leaving a security dragline [10]. With such reluctance to lose grasp of a solid substrate,
we find it much less likely that they would adventure in ballooning.
The other alternative to explain transoceaniclong-distance dispersal of this genus would
therefore be oceanic drift, or rafting, aided by the Antarctic Circumpolar Current and possibly
the West Wind Drift pushing floating matter along the ocean’s surface, as found for other
organisms, e.g. [7680]. Relatively few studies have postulated [2024,26] or even observed
[19,81] rafting in low-vagility spiders. While there are myriads of spider genera endemic to
high latitudes in the Southern Hemisphere, and many of them belong to excellent ballooning
families, Amaurobioides is, to our knowledge, the only one that inhabits South Africa, Austra-
lia, New Zealand and South America [24,82]. Another genus with a similar geographical pat-
tern is Desis (Desidae), but it extends into lower latitudes (Africa, southern India, South East
Asia and Japan, islands of the South Pacific Ocean including Australia and New Zealand, and
the Galapagos Islands). Both genera inhabit coastal zones and we believe that rafting is a more
plausible mechanism than ballooning to explain their distribution, agreeing with the hypothe-
sis presented by Hewitt [35]. This hypothesis is strengthened by the current findings on
Amaurobioides mirroring the patterns found in marine fauna also believed to have distributed
by rafting [83], their coastal habits, and proved ability to withstand immersion [29,84]. This
study therefore represents an exceptional case for spiders of a Southern Hemisphere distribu-
tion shaped by successful LongDistance Dispersal events by founder individuals/ populations
rafting with the Antarctic Circumpolar Current and the West Wind Drift.
This work adds to the literature showing that Amaurobioides is a remarkable anyphaenid
spider. Being the only genus of the sub-family with speciesfound outside the American conti-
nent, its adaptation to living in coastal habitats allowed for transoceanic dispersal events, pre-
sumably by rafting, to establish new species on the coasts of SouthernAfrica, Australia, New
Zealand and South America. Furthermore, the dispersal events are likely to have been in a pre-
dominantly eastward direction, fitting in timing and direction with the onset and continued
geo-climatic phenomena of the Antarctic Circumpolar Current and West Wind Drift. This
study therefore adds to the cases of dispersal asymmetry found in the Southern Hemisphere
[69,85], not only for well-known long-distance dispersers but also for organisms with poor
dispersal abilities of their own, such as Amaurobioides.
Supporting Information
S1 File. Table A. Information for tissuesamples and GenBank accession codesfor sequences.
Table B. Primers used for PCR. Table C. Partitioning scheme and nucleotide substitution models
for phylogenetic analyses. Table D. Estimates of net evolutionary divergence. Table E. Mean rates
for molecular markers. Table F. BioGeoBEARS outputs based on the BEAST species tree.
Table G. BioGeoBEARS outputs based on the concatenated BEAST tree. Matrix A. Uncon-
strained dispersal multiplier matrix. Matrix B. Distance constrained dispersal multiplier matrix.
Matrix C. East-to-west (EWD) constrained dispersal multiplier matrix. Matrix D. West-to-east
(WWD) constrained dispersal multiplier matrix. Figure A. Phylogenetic gene tree for COI.
Figure B. Phylogenetic gene tree for 16S. Figure C. Phylogenetic gene tree for H3a. Figure D.
Phylogenetic gene tree for 28S. Figure E. Chronogram inferred using the concatenated COI, 16S,
H3a and 28S data. Figure F. Species coalescence tree with node age estimates based COI, 16S, H3a
and 28S. Figure G. Ancestral range estimates based on the concatenated topology from BEAST.
Amaurobioides Long-Distance Eastward Dispersal
PLOS ONE | DOI:10.1371/journal.pone.0163740 October 12, 2016 15 / 20
New Zealand Field Work. Simon Pollard, the late Lyn Forster, Shirley McQueen, Ilana Batche-
lor, and Chris Green helped arrange collecting and export permits that were issued by New
Zealand’s Department of Conservation. Grace Hall provided alcohol for preserving spiders and
access to Landcare Research collections. Cor Vink and Simon Pollard organised collecting trips
to Bank’s Peninsula and Simon helped collect spiders at Tumbledown Bay; Peter Michalik and
Cor Vink also organised a collecting trip to South Island. Robin Andrews and Denis Gibbs
helped collect Amaurobioides.Australian Field Work. Leslie Chisholm at the South Australian
Museum and Elizabeth Turner at the Tasmanian Museum and Art Gallery provided advice,
helped with arrangements for fieldwork, and allowed BDO to examine specimens in their col-
lections. The South Australian Department of Environment and Natural Resources and the
Tasmanian Department of Primary Industries, Parks, Water, and Environment issued collect-
ing permits (with advice from Michael Driessen). Bronwyn Meredith of Australian Wildlife
Trade Assessments processed the export permit. Robin Andrews and Peter Michalik helped
collect Amaurobioides.South African specimens. Candice Owen (Rhodes University, Gra-
hamstown, South Africa) and Dawn Larsen (Iziko South African Museum, Cape Town, South
Africa) are thanked for providing fresh material for inclusion in this study. We also thank Cor
Vink and Miquel Arnedo for their constructive comments which greatly improved the
Author Contributions
Conceptualization:FSC MJR.
Data curation: FSC BDO CRH RJR EMS MJR.
Formal analysis: FSC MJR.
Funding acquisition: BDO CRH RJR MJR.
Investigation: FSC MJR.
Methodology: FSC MJR.
Project administration: MJR.
Supervision: MJR.
Validation: FSC MJR.
Visualization: FSC.
Writing – original draft: FSC.
Writing – review & editing: FSC BDO CRH RJR EMS MJR.
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Supplementary resource (1)

... Second, we found evidence for at least one trans-oceanic long-distance dispersal between South Africa and Mexico in the Eocene. Discoveries of such extreme trans-oceanic dispersals are becoming increasingly common 41,99,112 and can be attributed to salt-water tolerance 113 , long-distance flight [114][115][116] , and wind-dispersal in plants 99 and ballooning spiders 117,118 . Exchanges between Africa, the Western Indian Ocean, and the Neotropics have been especially well documented in tropical flora [119][120][121][122][123][124][125] . ...
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Net-casting spiders (Deinopidae) comprise a charismatic family with an enigmatic evolutionary history. There are 67 described species of deinopids, placed among three genera, Deinopis, Menneus, and Asianopis, that are distributed globally throughout the tropics and subtropics. Deinopis and Asianopis, the ogre-faced spiders, are best known for their giant light-capturing posterior median eyes (PME), whereas Menneus does not have enlarged PMEs. Molecular phylogenetic studies have revealed discordance between morphology and molecular data. We employed a character-rich ultra-conserved element (UCE) dataset and a taxon-rich cytochrome-oxidase I (COI) dataset to reconstruct a genus-level phylogeny of Deinopidae, aiming to investigate the group’s historical biogeography, and examine PME size evolution. Although the phylogenetic results support the monophyly of Menneus and the single reduction of PME size in deinopids, these data also show that Deinopis is not monophyletic. Consequently, we formally transfer 24 Deinopis species to Asianopis; the transfers comprise all of the African, Australian, South Pacific, and a subset of Central American and Mexican species. Following the divergence of Eastern and Western deinopids in the Cretaceous, Deinopis/Asianopis dispersed from Africa, through Asia and into Australia with its biogeographic history reflecting separation of Western Gondwana as well as long-distance dispersal events.
... Alternatively, island colonization by Dysdera has been suggested to be most likely mediated by rafting on plant and soil debris (Arnedo et al., 2001). Similarly, oceanic drifting was invoked to explain the Gondwanan distribution of Amaurobioides (Anyphaenidae) ghost spiders, which would have been favoured by the coastal habitat and the ability for immersion of these spiders, and the presence of the Antarctic Circumpolar Current and the West Wind Drift (Ceccarelli et al., 2016). Interestingly, the new species here described inhabit the spray zone of their respective islands. ...
The woodlouse hunter Dysdera spiders have colonized all Macaronesian archipelagos. We report here for the first time an evolutionary connection between the Iberian Peninsula, Madeira, and the remote archipelago of Azores. Based on museum specimens from the 1950s, we describe the first endemic Dysdera species from the Azores. Additionally, we report the recent collection of immature individuals related yet probably not conspecific to the new species, rejecting previous suggestions that the endemic lineage had gone extinct. A multi-locus target phylogeny revealed that an undescribed species from Madeira was the closest relative to the Azores lineage, and that both island taxa were in turn sister to an Iberian endemic species, within a mostly Iberian clade. Interestingly, the Madeiran relative was not closely related to the remaining endemic species reported in the archipelago, suggesting an independent colonization. A divergence time estimation analysis unravelled that Dysdera colonized both archipelagos early after their emergence. The colonization pathway remains ambiguous, but the Iberian Peninsula acted as the ultimate source of the ancestral colonizers. Finally, we describe the new species Dysdera cetophonorum Crespo & Arnedo sp. nov. from Pico and Dysdera citauca Crespo & Arnedo, sp. nov. from Ilhéu de Cima (Porto Santo) and redescribe and illustrate the female genitalia for the first time of their poorly known closest relative, Dysdera flavitarsis Simon, 1882 from the north-western Iberian Peninsula.
... Given that the Caribbean lineages of Micrathena have a North/Central American origin, the loop current, wrapping around the Gulf of Mexico, entering by the Yucatán peninsula, and exiting via the straights of Florida [91], may be of particular import as it brushes close to Greater Antillean islands. The long-distance dispersal, via rafting in arachnids, has been documented in Moggridgea mygalomorphs in Australia [92] and in Amaurobioides [93]. Paleocurrent directionality in the Caribbean, which most likely mirrors that of the Holocene (although a thruway between the Atlantic and Pacific existed before the closure of the Panama isthmus at 3.5 Ma) [94][95][96], and it can be hypothesized that the dispersal routes that allowed Micrathena to colonize the Caribbean reflect modern and paleooceanographic dynamics. ...
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Island biogeographers have long sought to elucidate the mechanisms behind biodiversity genesis. The Caribbean presents a unique stage on which to analyze the diversification process, due to the geologic diversity among the islands and the rich biotic diversity with high levels of island endemism. The colonization of such islands may reflect geologic heterogeneity through vicariant processes and/ or involve long-distance overwater dispersal. Here, we explore the phylogeography of the Caribbean and proximal mainland spiny orbweavers (Micrathena, Araneae), an American spider lineage that is the most diverse in the tropics and is found throughout the Caribbean. We specifically test whether the vicariant colonization via the contested GAARlandia landbridge (putatively emergent 33–35 mya), long-distance dispersal (LDD), or both processes best explain the modern Micrathena distribution. We reconstruct the phylogeny and test biogeographic hypotheses using a ‘target gene approach’ with three molecular markers (CO1, ITS-2, and 16S rRNA). Phylogenetic analyses support the monophyly of the genus but reject the monophyly of Caribbean Micrathena. Biogeographical analyses support five independent colonizations of the region via multiple overwater dispersal events, primarily from North/Central America, although the genus is South American in origin. There is no evidence for dispersal to the Greater Antilles during the timespan of GAARlandia. Our phylogeny implies greater species richness in the Caribbean than previously known, with two putative species of M. forcipata that are each single-island endemics, as well as deep divergences between the Mexican and Floridian M. sagittata. Micrathena is an unusual lineage among arachnids, having colonized the Caribbean multiple times via overwater dispersal after the submergence of GAARlandia. On the other hand, single-island endemism and undiscovered diversity are nearly universal among all but the most dispersal-prone arachnid groups in the Caribbean.
... While on the one hand recent divergence time estimation analyses have validated vicariance hypotheses (Bakkes et al., 2018;Jerome et al., 2014;Joshi & Edgecombe, 2019;Toussaint et al., 2016), on the other hand it could be shown that they have falsified vicariance explanations in cases showing that dispersals across even large barriers like oceans have taken place (McDowall, 2002;Raxworthy et al., 2002;Schrago & Russo, 2003;Yoder et al., 2003). Furthermore, longdistance dispersal has been demonstrated for certain cosmopolitan taxa with disjunct distributions that diverged only in the Tertiary (Ceccarelli et al., 2016) or that are considered less mobile (Rota et al., 2016;Trewick, 2000;Ward, 2014). Perhaps recent widespread distributions may be more dependent on a species' ability to colonize new niches than on dispersal ability. ...
Aim Cosmopolitan distributions have classically been explained by Pangaean vicariance. However, evidence of recently diverged cosmopolitan groups has re‐opened consideration on the processes involved. Our aim is to estimate the processes leading to the worldwide distribution of the kleptoparasitoid genus Ceropales. Location Worldwide. Taxon Ceropales spider wasps (Pompilidae). Methods Data from three molecular markers for 52 specimens of Ceropales and two calibration points from previous analyses were used to reconstruct a dated phylogeny under a relaxed molecular clock. We compared the fit of 12 models using BioGeoBEARS: DEC (subset sympatry, narrow vicariance), DIVALIKE (narrow and wide vicariance), BAYAREALIKE (widespread sympatry), and these same models with an added jump dispersal parameter and constraining dispersal rates among areas. Using the AIC best‐fit model (DEC+J), we performed Biogeographic Stochastic Mapping (BSM) to infer biogeographic processes by simulating 200 BSM on the BEAST chronogram. Results The origin of crown‐group Ceropales was ca. 10.6 Ma (15.7–6.5 95% HPD), and 11 jump‐dispersal events explain its distribution. A constrained DEC+J model, allowing adjacent area dispersal was the best‐fit AIC model. Dispersals across the Bering land bridge, Isthmus of Panama, Mediterranean Sea, and Sunda Plains took place from the late Miocene to present times. Main conclusions Ceropales is a recently diverged group that originated in Eurasia in the Miocene and dispersed to occupy the Americas, Africa, and Australia. Colonization was probably favored by the already diversified hosts (Pompilinae and Pepsinae), which reduced limiting factors such as food resource and nest construction. The evolution of a generalist parasitic lifestyle could facilitate long‐distance dispersal. This is the first study addressing the global historical biogeography of a cosmopolitan spider wasp.
... The Cladomorphinae of Central and South America also originated in Southeast Asia and can be interpreted as the result of a transoceanic, most probably trans-Pacific dispersal event, or via Antarctica which was connected to Australia and South America. Ancient longdistance dispersals in eastward direction across the Pacific have been reported for terrestrial arthropods before, for instance in Amaurobioides coastal spiders (Araneae; Ceccarelli et al., 2016) and metalmark moths (Lepidoptera: Choreutidae, Rota et al., 2016). Transoceanic crossings repeatedly played a pivotal role for stick and leaf insect distribution (Buckley et al., 2009(Buckley et al., , 2010, with oceanic distances covered as far as from Australia to the Mascarene Archipelago . ...
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Phasmatodea comprises over 3,000 extant species and stands out as one of the last remaining insect orders for which a robust, higher-level phylogenetic hypothesis is lacking. New research suggests that the extant diversity is the result of a surprisingly recent and rapid radiation that has been difficult to resolve with standard Sanger sequence data. In order to resolve the early branching events of stick and leaf insects, we analyzed transcriptomes from 61 species, including 38 Phasmatodea species comprising all major clades and 23 outgroup taxa, including all other Polyneoptera orders. Using a custom-made ortholog set based on reference genomes from four species, we identified on average 2,274 orthologous genes in the sequenced transcriptomes. We generated various sub-alignments and performed maximum-likelihood analyses on several representative datasets to evaluate the effect of missing data and matrix composition on our phylogenetic estimates. Based on our new data, we are able to reliably resolve the deeper nodes between the principal lineages of extant Phasmatodea. Among Euphasmatodea, we provide strong evidence for a basal dichotomy of Aschiphasmatodea and all remaining euphasmatodeans, the Neophasmatodea. Within the latter clade, we recovered a previously unrecognized major New World and Old World lineage, for which we introduce the new names Oriophasmata tax. nov. (“Eastern phasmids”) and Occidophasmata tax. nov. (“Western phasmids”). Occidophasmata comprise Diapheromerinae, Pseudophasmatinae, and Agathemera, whereas all remaining lineages form the Oriophasmata, including Heteropterygidae, Phylliinae, Bacillus, Lonchodidae (Necrosciinae + Lonchodinae), Clitumninae, Cladomorphinae, and Lanceocercata. We furthermore performed a divergence time analysis and reconstructed the historical biogeography for stick and leaf insects. Phasmatodea either originated in Southeast Asia or in the New World. Our results suggest that the extant distribution of Phasmatodea is largely the result of dispersal events in a recently and rapidly diversified insect lineage rather than the result of vicariant processes.
Aim Fossil data may be crucial to infer biogeographical history, especially in taxa with tropical trans-Pacific distributions. Here, we use extinct and extant trochanteriid flattened spiders to test hypotheses that could explain its trans-Pacific disjunct distribution, including a Boreotropical origin with a North Atlantic dispersal, an African origin with South Atlantic dispersal and an Eurasian origin with Bering Bridge route. Location World-wide. Taxon Trochanteriidae, Plator-Doliomalus-Vectius (PDV) clade. Methods MicroCT was used to collect morphological data from an undescribed Baltic amber fossil. These data were used with additional fossils and extant species in a total-evidence, tip-dated phylogenetic analysis. We tested different scenarios using constrained dispersal matrices in a Bayesian approach. An analysis with fossils pruned was also performed to explore how lack of fossil data might impact inferences of biogeographical process. Results The phylogenetic analyses allowed us to place the new fossil in the genus Plator. Analyses without fossils suggest an African origin with a dispersal to Asia from India and a South Atlantic dispersal to South America. When fossils are included, hypothesis-testing rejects this scenario and equally supports a Boreotropical and an Afro-European origin with a South Atlantic route and a dispersal to Asia from Europe. Main conclusions Biogeographical inferences of disjunctly distributed taxa should be interpreted with caution when fossils are not included. Although one alternative hypothesis was not completely rejected, results show that the Boreotropical hypothesis for the PDV clade could be a robust explanation for its actual distribution. This hypothesis is mostly overlooked in animal taxa and rigorous tests with other taxa with similar distributions may reveal that a Boreotropical origin is common. We discuss methodological approaches that could improve biogeographical tests using fossils as terminals.
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The diversity of the genus Tafana Simon, 1903 is poorly known in the Neotropical regions. In this work we provide a taxonomic review of the genus as well as a phylogenetic analysis. The ingroup of the analysis is composed of sixteen species of Tafana and the outgroup is composed of five representatives of Anyphaenidae. The sister-group recovered for Tafana is the clade Aysha + Xiruana, being supported by the embolic process on the male bulb. Two species groups within Tafana are herein proposed, the silhavyi group and the riveti group, based on two exclusive synapomorphies in the male bulb. We redescribe Tafana quelchi and present a description of the previously unknown female of Tafana silhavyi, both from Venezuela. In addition, we describe the first adult specimens of Tafana straminea. Twelve new species, along with several previously described species, are described, illustrated and mapped: T. riveti, T. straminea, T. quelchi, T. kunturmarqa sp. nov., T. humahuaca sp. nov., T. pastaza sp. nov., T. nevada sp. nov., T. huatanay sp. nov. and T. ruizi sp. nov. from the riveti species group; T. maracay sp. nov., T. arawak sp. nov., T. chimire sp. nov. and T. pitieri sp. nov. from the silhavyi species group; T. oliviae sp. nov. from Argentina and T. orinoco sp. nov. from Venezuela, neither of which belongs to any species group. We also discuss the genital morphology of the species groups based on the results of the phylogenetic analysis. Furthermore, distribution maps for all species, including new records for T. riveti, T. straminea and T. quelchi, are presented.
Plocamium Lamouroux is a widespread genus for which 45 species are currently recognized. However, classical taxonomy based only on morphological characters, is problematic within this genus. The use of molecular tools has uncovered cryptic genetic species, mistakenly grouped under the name of morphological species that are common and widespread (including the generitype Plocamium cartilagineum (Linnaeus) P.S.Dixon). The aim of this work was to evaluate the species diversity of Plocamium in Southern Chile. For this purpose, three independent molecular markers were sequenced in samples collected from seven populations located between 41°S and 54°S. The species diversity was evaluated using phylogenetic reconstructions and two independent methods for species delimitation (ABGD and GMYC). The outcomes of each method were congruent, suggesting the presence of three species in Southern Chile. One species, named Plocamium sp. 1, is restricted to Punta Guabún, the only locality sampled north of the biogeographic barrier of the 42°S. The other two species, Plocamium sp. 2 and 3 are distributed in sympatry in Patagonia and Tierra del Fuego. The three Chilean species form a clade phylogenetically close to sequences obtained from New Zealand and Australia and a divergence along the coasts of Chile after past transoceanic dispersal is proposed. We propose that divergence in glacial microrefugia could have subsequently happen in the southern part of the coast, this hypothesis being supported by the strong impact of glacial maxima on population dynamics, especially in Plocamium sp. 3
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Few spider species show truly cosmopolitan distributions. Among them is the banded garden spider Argiope trifasciata that is reported from six continents across major climatic gradients and geographical boundaries. In orbweb spiders, such global distributions may be a result of lively dispersal via ballooning. However, wide distributions may also be artefactual owing to our limited understanding of species taxonomy. To test the hypothesis that A. trifasciata may be a complex of cryptic species with more limited geographic ranges, we here investigate the biogeographical structure and evolutionary history of A. trifasciata through a combination of time-calibrated phylogenetic analyses (57 terminals, 3 genes), ancestral range reconstruction, and species delimitation methods. Our results strongly suggest that A. trifasciata as currently defined is not a single species. Its populations fall into five reciprocally monophyletic clades that are genetically distinct and have evolutionary origins in the Plio – Pleistocene. These clades are confined to East Asia, temperate Australia, Hawaii, the New World, and the Old-World (Africa and most of the Palearctic). Our results provide the basis for a future investigation of morphological and/or ecological disparity between the populations that likely represent species, as well as examinations of these species’ attributes and dispersal modes.
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Studies in evolutionary biology and biogeography increasingly rely on the estimation of dated phylogenetic trees using molecular clocks. In turn, the calibration of such clocks is critically dependent on external evidence (i.e. fossils) anchoring the ages of particular nodes to known absolute ages. In recent years, a plethora of new fossil spiders, especially from the Mesozoic, have been described, while the number of studies presenting dated spider phylogenies based on fossil calibrations increased sharply. We critically evaluate 44 of these studies, which collectively employed 67 unique fossils in 180 calibrations. Approximately 54% of these calibrations are problematic, particularly regarding unsupported assignment of fossils to extant clades (44%) and crown (rather than stem) dating (9%). Most of these cases result from an assumed equivalence between taxonomic placement of fossils and their phylogenetic position. To overcome this limitation, we extensively review the literature on fossil spiders, with a special focus on putative synapomorphies and the phylogenetic placement of fossil species with regard to their importance for calibrating higher taxa (families and above) in the spider tree of life. We provide a curated list including 41 key fossils intended to be a basis for future estimations of dated spider phylogenies. In a second step, we use a revised set of 23 calibrations to estimate a new dated spider tree of life based on transcriptomic data. The revised placement of key fossils and the new calibrated tree are used to resolve a long‐standing debate in spider evolution – we tested whether there has been a major turnover in the spider fauna between the Mesozoic and Cenozoic. At least 17 (out of 117) extant families have been recorded from the Cretaceous, implying that at least 41 spider lineages in the family level or above crossed the Cretaeous–Paleogene (K–Pg) boundary. The putative phylogenetic affinities of families known only from the Mesozoic suggest that at least seven Cretaceous families appear to have no close living relatives and might represent extinct lineages. There is no unambiguous fossil evidence of the retrolateral tibial apophysis clade (RTA‐clade) in the Mesozoic, although molecular clock analyses estimated the major lineages within this clade to be at least ∼100 million years old. Our review of the fossil record supports a major turnover showing that the spider faunas in the Mesozoic and the Cenozoic are very distinct at high taxonomic levels, with the Mesozoic dominated by Palpimanoidea and Synspermiata, while the Cenozoic is dominated by Araneoidea and RTA‐clade spiders.
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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from free of charge.
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According to our unpublished data some 30 genera and 200 species of Salticidae can be expected in New Zealand. The fauna is highly endemic, both on a generic and a specific levels. The most diverse are two groups of genera: Trite minax [=planiceps] and "Trite" auricoma are the best known representatives of every group. The relationships between Salticidae of New Zealand and Australia, are limited to single representatives of Opisthoncus, Holoplatys, Ocrisiona, Helpis, "Lycidas", "Clynotis" and Hypoblemum. Wide-spread genera are represented by Neon and Bianor and pantropical Hasarius adansoni is found in the warmer climate of North Island. To a limited extent New Zealand is a source of fauna for other Pacific archipelagos, for example species of Trite are found in New Caledonia and Caroline Islands.
At a time when historical biogeography appears to be again expanding its scope after a period of focusing primarily on discerning area relationships using cladograms, new inference methods are needed to bring more kinds of data to bear on questions about the geographic history of lineages. Here we describe a likelihood framework for inferring the evolution of geographic range on phylogenies that models lineage dispersal and local extinction in a set of discrete areas as stochastic events in continuous time. Unlike existing methods for estimating ancestral areas, such as dispersal-vicariance analysis, this approach incorporates information on the timing of both lineage divergences and the availability of connections between areas (dispersal routes). Monte Carlo methods are used to estimate branch-specific transition probabilities for geographic ranges, enabling the likelihood of the data (observed species distributions) to be evaluated for a given phylogeny and parameterized paleogeographic model. We demonstrate how the method can be used to address two biogeographic questions: What were the ancestral geographic ranges on a phylogenetic tree? How were those ancestral ranges affected by speciation and inherited by the daughter lineages at cladogenesis events? For illustration we use hypothetical examples and an analysis of a Northern Hemisphere plant clade (Cercis), comparing and contrasting inferences to those obtained from dispersal-vicariance analysis. Although the particular model we implement is somewhat simplistic, the framework itself is flexible and could readily be modified to incorporate additional sources of information and also be extended to address other aspects of historical biogeography.
Amaurobioides are restricted to the spray zone of southern continents, where they live in small, isolated populations and hunt from silk retreats built in rock crevices. A Star BEAST species tree based on ITS1 nuclear and ND1 mitochondrial genes did not support the hypothesis that this unusual niche linked the evolutionary history of these spiders to geological events reshaping Gondwana into present-day Australia and New Zealand. Instead, it showed that Amaurobioides reached Australia approximately 4.5 Mya and dispersed twice to New Zealand. Approximately 2.37 Mya, spiders from Tasmania colonized the Deep South of the South Island and, approximately 0.38 Mya, those from South Australia colonized more northern regions. Thus, the present study further limits the scope of the Moa's Ark hypothesis of vicariant New Zealand biogeography.