Access to this full-text is provided by The Royal Society.
Content available from Proceedings of the Royal Society B
This content is subject to copyright.
rspb.royalsocietypublishing.org
Research
Cite this article: Ferna
´ndez R, Sharma PP,
Tourinho AL, Giribet G. 2017 The Opiliones tree
of life: shedding light on harvestmen
relationships through transcriptomics.
Proc. R. Soc. B 284: 20162340.
http://dx.doi.org/10.1098/rspb.2016.2340
Received: 25 October 2016
Accepted: 27 January 2017
Subject Category:
Evolution
Subject Areas:
evolution, molecular biology, genomics
Keywords:
Eupnoi, Dyspnoi, Cyphophthalmi, Laniatores,
phylogenomics, Arachnida
Authors for correspondence:
Rosa Ferna
´ndez
e-mail: rfernandezgarcia@g.harvard.edu
Gonzalo Giribet
e-mail: ggiribet@g.harvard.edu
Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.fig-
share.c.3691975.
The Opiliones tree of life: shedding light
on harvestmen relationships through
transcriptomics
Rosa Ferna
´ndez1, Prashant P. Sharma2, Ana Lu
´cia Tourinho1,3
and Gonzalo Giribet1
1
Museum of Comparative Zoology, Department of Organismic and Evolutionary Biology, Harvard University,
26 Oxford Street, Cambridge, MA 02138, USA
2
Department of Zoology, University of Wisconsin-Madison, 352 Birge Hall, 430 Lincoln Drive, Madison,
WI 53706, USA
3
Instituto Nacional de Pesquisas da Amazo
ˆnia, Coordenac¸a
˜o de Biodiversidade (CBIO), Avenida Andre
´Arau
´jo,
2936, Aleixo, CEP 69011-970, Manaus, Amazonas, Brazil
RF, 0000-0002-4719-6640; GG, 0000-0002-5467-8429
Opiliones are iconic arachnids with a Palaeozoic origin and a diversity that
reflects ancient biogeographic patterns dating back at least to the times of
Pangea. Owing to interest in harvestman diversity, evolution and biogeogra-
phy, their relationships have been thoroughly studied using morphology
and PCR-based Sanger approaches to infer their systematic relationships.
More recently, two studies utilized transcriptomics-based phylogenomics
to explore their basal relationships and diversification, but sampling was
limiting for understanding deep evolutionary patterns, as they lacked
good taxon representation at the family level. Here, we analysed a set of
the 14 existing transcriptomes with 40 additional ones generated for this
study, representing approximately 80% of the extant familial diversity in
Opiliones. Our phylogenetic analyses, including a set of data matrices
with different gene occupancy and evolutionary rates, and using a multitude
of methods correcting for a diversity of factors affecting phylogenomic data
matrices, provide a robust and stable Opilionestree of life, where most families
and higher taxa are precisely placed. Our dating analyses using alternative
calibration points, methods and analytical parameters provide well-resolved
old divergences, consistent with ancient regionalization in Pangea in some
groups, and Pangean vicariance in others. The integration of state-of-the-art
molecular techniques and analyses, together with the broadest taxonomic
sampling to date presented in a phylogenomic study of harvestmen, provide
new insights into harvestmen interrelationships, as well as an overview of
the general biogeographic patterns of this ancient arthropod group.
1. Introduction
Opiliones (‘harvestmen’ or ‘daddy longlegs’) are a remarkable group of ara-
chnids (electronic supplementary material, figure S1), with a fossil record
dating to the Early Devonian, having diversified in its main lineages by the
Carboniferous [1– 3], and showing ancient vicariant patterns that accord with
their modern distribution [4 – 8]. They show fascinating reproductive beha-
viours, including paternal and biparental care [9– 12], and constitute an
example of the first direct transfer of sperm on land via a penis [1].
The phylogeny of the order Opiliones and its four extant suborders—
Cyphophthalmi (the mite harvestmen), Eupnoi (the daddy longlegs), Dyspnoi
(the ornate harvestmen) and Laniatores (the armoured harvestmen)—has
received considerable attention, based on morphological [13– 17], molecular
[18–21] and combined datasets [3,22,23]. After some debate, the relationships
&2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
author and source are credited.
among the Opiliones suborders have been settled, with
Cyphophthalmi constituting the sister group of Phalangida,
the latter divided in Palpatores (Eupnoi þDyspnoi) and
Laniatores. More recently, a few studies have used phyloge-
nomic data derived from transcriptomes to further test
relationships among Opiliones [24– 26], but these pioneering
studies included a handful of species (8–14) representing just
a few families. Likewise, the internal relationships of each of
the four suborders have received attention, mostly using mol-
ecular [4,20,27– 29] and combined analyses of morphology and
molecules [8]. Other morphological analyses have focused on
particular suborders [30–33]. Recently, a Dyspnoi cladogram
was proposed based on a summary of proposed relationships
[34]. In addition, dozens of papers have explored the relation-
ships of individual families or groups of closely related species.
While many aspects of the phylogeny of Opiliones are
now well understood, a few remain largely unresolved or
understudied. For example, within Cyphophthalmi, the
relationships among its six families, and even the monophyly
of Sironidae, remain unsettled [8]. Relationships within
Eupnoi—the group that includes the true ‘daddy longlegs’—
are barely explored from a molecular perspective [20,35,36],
and no study has included all the relevant diversity. Resolution
within these clades is poor, with the exception of the deepest
division between Caddoidea and Phalangioidea [20]. Relation-
ships within Dyspnoi are just beginning to settle [28,29,37], but,
for example, only recently was it recognized that Acropsopilio-
nidae are related to Dyspnoi and not to Eupnoi [20], based on a
handful of Sanger-sequenced molecular markers. This resulted
in transferring aclade of Opiliones from Eupnoi to Dyspnoi, as
the sister group to all other members (Ischyropsalidoidea þ
Troguloidea), and therefore deserves further testing using a
modern and more complete dataset. Finally, relationships
within Laniatores have changed considerably after the study
of Sharma & Giribet [27], as the taxonomy of this large clade
of Opiliones has been in flux, with description of several
families in recent years [27,38– 40]. Some novel results include
the proposal of a sister group relationship of the New Zealand
endemic family Synthetonychiidae to all other Laniatores
[19,27]—a result that hinged on partial data from a single
species. In addition, the relationships among many families
remain unstable.
Recent application of dense taxon sampling using large
numbers of genes through modern phylogenomic approaches
(e.g. based on genome and Illumina-based datasets) has
resolved family-level relationships of a diversity of groups of
arachnids [41–44] and other arthropods [45,46]. We applied
these methodologies to Opiliones phylogenetics to produce a
densely sampled family-level phylogeny by analysing 54 har-
vestman transcriptomes (40 newly generated for this study
and 14 previously published) representing 40 of the 50 cur-
rently recognized extant families (80% familial representation).
2. Material and methods
(a) Specimens
Specimens of Opiliones selected for this study were preserved in
RNAlater and transferred to liquid nitrogen upon arrival to the
laboratory, or flash-frozen, and subsequently stored at 2808C.
Total RNA, mRNA purification and library construction proto-
cols are explained in detailed in the electronic supplementary
material, Extended material and methods and S1).
Our final matrix comprises 54 taxa, including 10 Cypho-
phthalmi (four families included; Ogoveidae and Troglosironidae
missing), nine Eupnoi (representatives of all five families included),
nine Dyspnoi (all eight families included), and 26 Laniatores
(representatives of 23 families included; missing Gerdesiidae,
Guasiniidae, Icaleptidae, Kimulidae, Metasarcidae, Nipponony-
chidae, Pyramidopidae and Tithaeidae, all families of low
diversity and relatively narrow distribution in places difficult to
access). As outgroups, we included several chelicerates (see elec-
tronic supplementary material, Extended material and methods
and table S1).
All raw sequences are deposited in the SRA archive of
GenBank under accession numbers specified in electronic sup-
plementary material, table S1. Data on specimens are available
from MCZbase (http://mczbase.mcz.harvard.edu).
(b) Orthology assignment and phylogenetic analyses
Orthology assignment was based on the OMA algorithm v. 0.99.z3
[47], as specified in detail in our previous work [48]. Multiple
sequence alignment, alignmentmasking and criteria for matrix con-
struction followed our previous workflows [48] and are detailed in
the electronic supplementary material. Maximum-likelihood infer-
ence was conducted with PHYML-PCMA [49], EXAML [50] and
PHYML v. 3.0.3. Bayesian analyses were conducted with EXABAYES
[51] and PHYLOBAYES MPI 1.4e [52] using the site-heterogeneous
CAT-GTR model of evolution in the latter software [53]. Compo-
sitional homogeneity of each gene and taxon was evaluated in
BACOCA[54]. Details on the different matrices, priors and heuristics
are catalogued in the electronic supplementary material.
(c) Molecular dating
The fossil record of Opiliones is well documented, and most key
fossils have been included in prior phylogenetic analyses,
making their placement in a phylogenetic context precise. We
mostly follow the strategy and fossil placement of Sharma &
Giribet [25], who conducted tip dating in one of their analyses.
Exact details about the fossils selected and the type of constraints
used are described in the electronic supplementary material,
Extended material and methods.
Divergence dates were estimated using the Bayesian relaxed
molecular clock approach as implemented in PHYLOBAYES v. 3.3f
[52] under the autocorrelated lognormal and uncorrelated
gamma multipliers models, resulting in four analyses (i.e. these
two models were applied to both calibration configurations
described above, with the age of Eophalangium as the minimum
age of Cyphophthalmi or as the floor of Opiliones). Two indepen-
dent MCMC chains were run for each analysis (10 000 –12 000
cycles). The calibration constraints were used with soft bounds
[55] under a birth–death prior.
3. Results and discussion
All results are based on three original data matrices of 78
genes (matrix I; more than 90% gene occupancy), 305 genes
(matrix II; more than 75% gene occupancy) and 1550 genes
(matrix III; more than 50% gene occupancy), as well as sub-
sets of these matrices (see Material and methods). Figure 1
(see also electronic supplementary material, figure S2) illus-
trates the topology obtained for matrix I in PHYML_PCMA,
with a Navajo rug representing the support for the 16
analyses conducted for the different matrices and methods.
(a) Higher-level Opiliones phylogenetics
Our analyses recover a stable relationship among the four extant
Opiliones suborders, each well supported as monophyletic in
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
2
all the analyses (figure 1), as consistently found in a variety
of published Opiliones analyses (e.g. [16,18,19,20,24–26]),
including phylogenomic ones [24– 26]. Likewise, we found
Cyphophthalmi as sister group to Phalangida, monophyly of
Palpatores, and a sister group relationship of Palpatores to
Laniatores, as in nearlyall recent studies cited above. However,
most published analyses found little support for the resolution
within each suborder—in Sanger-based analyses due to insuffi-
cient sequence data and in phylogenomic analyses due to few
taxa. The resolved relationships within each of the four subor-
ders are thus the most novel aspects of this study. Each
suborder is therefore discussed in detail below.
(b) Cyphophthalmi—the mite harvestmen
The members of the suborder Cyphophthalmi (electronic
supplementary material, figure S1a) have received special
attention phylogenetically due to their antiquity, their global
distribution and their low vagility (e.g. [4,8]). Here, we confirm
the division of Cyphophthalmi into the temperate Gondwanan
family Pettalidae and the remaining families (Stylocellidae,
Neogoveidae, Sironidae) (figure 1), a divergence that took
place around the Jurassic, diversifying during the Cretaceous
(figure 2). While the New Caledonian endemic
Troglosironidae and the west African endemic Ogoveidae
were not included, their phylogenetic affinity to Neogoveidae
in the clade Sternophthalmi is strongly supported by an
array of morphological and molecular datasets [8].
Relationships among Stylocellidae, Neogoveidae and
Sironidae are unstable, and two topologies prevail: (Stylocelli-
dae, (Neogoveidae, Sironidae)) and (Sironidae, (Stylocellidae,
Neogoveidae)), neither topology supporting the taxon Bor-
eophtahlmi, grouping Stylocellidae and Sironidae [8]
(figure 1; electronic supplementary material, figure S2).
The first topology is preferred by the most complete dataset.
However, in some of the analyses using fewer genes the
0.08
Synsphyronus apimelus
Aoraki longitarsa
Homalenotus remyi
Pseudopachylus longipes
Nipponopsalis abei
Metagovea oviformis
Odiellus troguloides
Leiobunum verrucosum
Sitalcina lobata
Zalmoxis gebeleizis
Saramacia lucasae
Escadabius n. sp.
Protimesius longipalpis
Ischyropsalis nodifera
Dibunus sp.
Idzubius akiyamae
Fissiphallius martensi
Tetranychus urticae
Parasiro coiffati
Pseudocellus pearsei
Dicranolasma soerenseni
Caddo pepperella
Fumontana deprehendor
Sclerobunus robustus
Metasiro americanus
Brasilogovea microphaga
Anoplodactylus insignis
Siro americanus
Gyas titanus
Phalangium opilio
Petrobunus schwendingeri
Dampetrus sp.
Phareicranaus manauara
Neopurcellia salmoni
Liphistius malayanus
Acropsopilio neozealandiae
Protolophus singularis
Vonones ornata
Pellobunus sp.
Trogulus martensi
Ortholasma coronadense
Theromaster sp.
Hesperonemastoma modestum
Rakaia magna australis
Stylocellidae sp.
Nemastomella dubia
Eremobates sp.
Ricinoides atewa
Thrasychirus gulosus
Metagyndes innata
Chapulobunus unispinosus
Avima matintaperera
Daphnia pulex
Suzukielus sauteri
Limulus polyphemus
Metabiantes sp.
Synthetonychia glacialis
Metibalonius sp.
Gnomulus sp.
Sabacon cavicolens
Centruroides sculpturatus
Scutigera coleoptrata
Forsteropsalis pureora
Pachylicus acutus
Ixodes scapularis
gf
p
St
y
locellidae
sp
.
y
Odiellus troguloide
s
Caddo pepperella
Gy
as titanu
s
Phalan
g
ium opili
o
Protolophus sin
g
ularis
Pseudopach
y
lus lon
g
ipe
s
S
italcina lobat
a
Zalmoxis
g
ebeleizi
s
Saramacia lucasa
e
Es
ca
d
abiu
s
n. s
p
.
Protimesius lon
g
ipalpi
s
Dibunus
sp
.
Idzubius akiyamae
F
issi
p
hallius martensi
F
umontana deprehendor
S
clerobunus robustu
s
Petrobunus schwendin
g
eri
Dam
p
etrus
sp
.
Phareicranau
s
manauar
a
V
onones ornata
Pellobunu
s
sp
.
Theromaster
sp
.
Meta
gy
ndes innat
a
Chapulobunus unispinosu
s
Avima matintaperer
a
Metabiante
s
sp
.
Sy
ntheton
y
chia
g
laciali
s
Metibaloniu
s
s
p.
G
nomulus
sp
.
y
Pach
y
licus acutu
s
Ni
pp
ono
p
salis abei
Ischyropsalis nodi
f
er
a
Dicranola
s
ma
s
oeren
s
eni
Acro
p
so
p
ilio neozealandiae
Tro
g
ulus martensi
Hes
p
eronemastoma modestu
m
S
abacon ca
v
icolen
s
Aora
k
i
l
ongitars
a
Neo
p
urcellia salmoni
Ra
k
aia magna austra
l
i
s
Meta
g
ovea ovi
f
ormi
s
Meta
s
iro americanu
s
Brasilo
g
ovea micropha
ga
Parasiro coi
ff
at
i
S
iro americanu
s
S
uzu
k
ie
l
us sauter
i
PETTALIDAE
STYLOCELLIDAE
NEOGOVEIDAE
SIRONIDAE
CYPHOPHTHALMI
EUPNOI
DYSPNOI LANIATORES
ppp
T
hras
y
chirus
g
ulosu
s
F
orstero
p
salis
p
ureora
Homalenotus rem
yi
Leiobunum verrucosu
m
CADDIDAE
NEOPILIONIDAE
PROTOLOPHIDAE
SCLEROSOMATIDAE
PHALANGIIDAE
TAR AC ID AE
ISCHYROPSALIDIDAE
SABACONIDAE
O
rtholasma coronadens
e
N
emastomella dubia NEMASTOMATIDAE
NIPPONOPSALIDIDAE
TROGULIDAE
DICRANOLASMATIDAE
TRIAENONYCHIDAE
SYNTHETONYCHIIDAE
CLADONYCHIIDAE
TRAVUNIIDAE
Peripatopsis overbergiensis
PHALANGODIDAE
SANDOKANIDAE
EPEDANIDAE
PETROBUNIDAE
PODOCTIDAE
ASSAMIIDAE
SAMOIDAE
BIANTIDAE
ESCADABIIDAE
FISSIPHALLIIDAE
ZALMOXIDAE
STYGNOPSIDAE
AGORISTENIDAE
STYGNIDAE
CRYPTOGEOBIIDAE
COSMETIDAE
GONYLEPTIDAE
MANAOSBIIDAE
CRANAIDAE
I II III IV V VI
EB EM EB EM
EB
EM
EB
PB PB
PP
PB
PB
PB
PP
PIL
PIL
*
ACROPSOPILIONIDAE
node with low support
node not recovered
(>0.95 PP, >90% BS)
*(0.90 > 0.95 PP,
80 > 90% BS)
*
**
Insidiatores
Phalangodoidea
Sandokanoidea
Epedanoidea
Assamioidea
Samooidea/Zalmoxoidea
Gonyleptoidea
Larifuga capensis
Figure 1. Phylogenetic hypothesis based on the 78-gene matrix I analysed in PHYML_PCMA (2lnL¼2248960.37) Selected deep nodes (grey circle) show Navajo
rug illustrating support under specific data matrices and analyses. In Laniatores, coloured text for family names indicates superfamily boundaries. EB: EXABAYES. EM:
EXAML. PB: PHYLOBAYES. PIL: PHYML with integrated branch lengths. PP: PHYML-PCMA. (Online version in colour.)
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
3
Stylocellidae species nests within Sironidae, albeit without
support. These two alternatives will require further examin-
ation with more stylocellid samples, as the alternative
topologies may also have an impact on the dating, which
suggests an initial diversification around the Cretaceous
(figure 2).
Monophyly of Neogoveidae is recovered in all analyses,
with the exception of the PHYLOBAYES analysis of the 78-
gene matrix (electronic supplementary material, figure S2).
The placement of the North American Metasiro with the typi-
cal Neotropical neogoveids corroborates previous molecular
hypotheses of the delimitation of this clade [8,56].
Monophyly of Sironidae, here represented by three
genera of the three main lineages of this Laurasian family
(Siro,Parasiro and Suzukielus), is unstable across analyses
(electronic supplementary material, figure S2). Monophyly
of Sironidae has been difficult to obtain in molecular analyses
as well as with morphology due to differences in family-level
characters in the western Mediterranean Parasiro and the
Japanese Suzukielus [8].
(c) Eupnoi—the daddy longlegs
Family-level Eupnoi phylogenies are scarce [8,20,35] and
have typically undersampled Southern Hemisphere lineages.
Our analyses support the well-known division of Caddoidea
(electronic supplementary material, figure S1b) and Phalan-
gioidea (electronic supplementary material, figure S1c,d),
which in turn divides into the Southern Hemisphere
Neopilionidae and the mostly Northern Hemisphere families
Phalangiidae, Sclerosomatidae and Protolophidae—although
Phalangiidae and Sclerosomatidae have later diversified in
the Southern Hemisphere (figure 1; electronic supplementary
material, figure S2). The sister group relationship among
0
100200300400
Parasiro
Aoraki
Neopurcellia
Siro
Stylocellidae
Metasiro
Brasilogovea
Rakaia
Metagovea
Suzukielus
My
P
ara
si
ro
Aoraki
N
eopurce
ll
i
a
S
ir
o
S
t
y
locellidae
M
eta
s
iro
B
rasi
l
o
g
ovea
R
a
k
aia
M
eta
g
ovea
S
uzukielu
s
Nipponopsalis
Phalangium
Ortholasma
Protolophus
Ischyropsalis
Homalenotus
Sabacon
Odiellus
Nemastomella
Caddo
Dicranolasma
Hesperonemastoma
Acropsopilio
Gyas
Thrasychirus
Forsteropsalis
Leiobunum
Trogu lu s
Aoraki
Ph
a
l
angiu
m
P
roto
l
op
h
u
s
H
oma
l
enotu
s
O
diellus
C
a
dd
o
G
yas
Th
rasyc
h
iru
s
F
orsteropsa
l
i
s
L
eio
b
unu
m
N
i
pp
ono
p
sali
s
O
rt
h
o
l
asm
a
I
sc
h
yropsa
l
is
Sa
b
acon
N
emastome
ll
a
D
icrano
l
asma
H
esperonemastom
a
A
cro
p
so
p
i
l
i
o
Trogu
l
us
20
0
2
40
0
4
30
0
0
Early Devonian 390 Ma Late Carboniferous 306 Ma Early Jurassic 195 Ma
Escadabius
Pachylicus
Fissiphallius
Dampetrus
Phareicranaus
Pseudopachylus
Pellobunus
Dibunus
Protimesius
Saramacia
Avima
Sclerobunus
Von o n e s
Gnomulus
Larifuga
Metibalonius
Fumontana
Chapulobunus
Zalmoxis
Metagyndes
Sitalcina
Idzubius
Metabiantes
Synthetonychia
Theromaster
Petrobunus
Late Cretaceous 94 Ma
CYPHOPH.
EUPNOIDYSPNOI
LANIATORES
Figure 2. (a) Chronogram of Opiliones evolution for the 78-gene dataset with 95% highest posterior density (HPD) values for the dating for the first calibration
configuration (i.e. the age of Eophalangium as the minimum age of Cyphophthalmi) under uncorrelated gamma model. Down, palaeogeographical reconstruction
according to Christopher R. Scotese (maps modified from http://www.scotese.com/earth.htm) at some of the key ages of the split of Opiliones main lineages, as
recovered by the molecular dating analysis. Vertical bars indicate correspondence with each palaeomap following a colour code. (Online version in colour.)
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
4
Protolophidae and Sclerosomatidae has been found in pre-
vious analyses [20,35], and in fact some have considered
Protolophidae a junior synonym of Sclerosomatidae [57].
However, resolution among the families of Phalangioidea
has received little or no support in previous studies. Our
results thus provide, for the first time, a well-resolved
Eupnoi phylogeny, including the placement of Gyas titanus
within Phalangiidae, as suggested by Hedin et al. [35],
instead of within Sclerosomatidae. We were not able to
include any members of the phylogenetically unstable ‘Meto-
pilio group’ [19,35]. We thus find Phalangioidea divided into
three main clades: Neopilionidae, Sclerosomatidae/Protolo-
phidae and Phalangiidae (including Gyas). However, the
systematics of this large group of Opiliones, with nearly
200 genera and 1800 species, will require much denser
sampling before the group can be properly revised.
(d) Dyspnoi—the ornate harvestmen
The global phylogeny of Dyspnoi has received attention from
different workers using morphology and molecules, but only
recently there has been modern treatment. Groh & Giribet
[20] finally circumscribed the suborder, transferring
Acropsopilionidae from Eupnoi to the sister group of all
other Dyspnoi based on molecular data analyses of a few
Sanger-sequencing genes and morphological examination.
Our phylogenomic datasets corroborate this topology
(figure 1), placing Acropsopilio neozealandiae as the sister
group to the other Dyspnoi, with the monophyly of each of
Ischyropsalidoidea and Troguloidea being fully supported.
While the position of the Cretaceous fossil Halitherses grimal-
dii remains uncertain, their large eyes (resembling those of
caddids and acropsopilionids) and their troguloid facies [58]
suggest a phylogenetic placement between Acropsopilionoi-
dea and the remaining Dyspnoi [59], perhaps as sister group
to Troguloidea or to Troguloidea þIschyropsalidoidea. How-
ever, the ‘caddoid’ gestalt is now known from Caddoidea,
Phalangioidea (in the members of the genus Hesperopilio;see
[20]) and Acropsopilionoidea, and enlarged eyes are thus
best optimized as a symplesiomorphy of Palpatores.
(e) Laniatores—the armored harvestmen
The phylogeny of Laniatores—the largest suborder of Opi-
liones with more than 4200 described species—has received
recent attention at many levels [19,27,60,61]. In an unpub-
lished thesis, Kury [62] divided Laniatores into Insidiatores
(electronic supplementary material, figure S1h–i) and
Grassatores (electronic supplementary material, figure S1j–p),
a division found here, but not in other studies that included
a meaningful sampling of Laniatores [19,27]. These found the
New Zealand endemic Synthetonychiidae to be sister group
to all other Laniatores (Eulaniatores sensu Kury [63]). Of special
interest also was the phylogenetic position of the unstable
North American Fumontana deprehendor, a member of the
mostly temperate Gondwanan family Triaenonychidae that
was poorly resolved in prior studies. Our analyses do find a
sister group relationship of Fumontana to the representative
of the Southern Hemisphere Triaenonychidae in virtually all
analyses, with a Cretaceous divergence (figure 2). Synthetony-
chia is either sister group to the represented travunioids in
most analyses (except for matrices IV and V; see electronic
supplementary material, figure S2) or sister group to Triaeno-
nychidae, as originally proposed by Forster [64]. Further
discussion on Insidiatores will require increased diversity of
genera both within Triaenonychidae and within the travunioid
families (see for example [57]).
Resolution within Grassatores has remained elusive except
for the recognition of a main division between Phalangodidae
and the remaining Grassatores and of the superfamilies
Gonyleptoidea, Assamioidea, Zalmoxoidea and Samooidea,
and perhaps a clade of southeast Asian families, Epedanoidea
[27]. Some of these clades were not supported in re-analyses of
the Sharma & Giribet dataset [60,61]. Here, we consistently
find Phalangodidae (represented by Sitalcina lobata)tobethe
sister group of the remaining Grassatores.
The southeast Asian endemic Sandokanidae [65] is
resolved as the sister group to the remaining families, a
clade supported by nearly all matrices and most analyses
(only some analyses find this clade without support)
(figure 1). The position of Sandokanidae has been difficult to
resolve in prior analyses [19,27], which sometimes suggested
a relationship to Epedanoidea. Here, we reject this hypothesis
and support Sandokanidae as the second offshoot of the Grass-
atores, contradicting earlier hypotheses dividing Grassatores in
Oncopodoidea versus Gonyleptoidea (e.g. [13,15]).
The sister group of Sandokanidae divides into the largely
southeast Asian Epedanoidea (represented here by members
of Epedanidae, Petrobunidae and Podoctidae) and a clade
including Assamioidea, Samooidea, Zalmoxoidea and Gony-
leptoidea, this divergence being Cretaceous (figure 2;
electronic supplementary material, figure S3). This coincides
with the first Laniatores fossils, which were already present
in the terranes of today’s Myanmar [66]. Epedanoidea is
monophyletic in all analyses (sometimes without significant
support), except for the PHYLOBAYES analysis of matrix VI
(electronic supplementary material, figure S2), and it is
resolved with Epedanidae being sister group to a clade of Pet-
robunidae and Podoctidae (electronic supplementary
material, figure S2; see also [61]). Resolving this may require
additional taxa, including the missing family Tithaeidae.
Epedanoidea has however been difficult to recover in a
recent analysis focusing on Podoctidae [61].
The sister group of Epedanoidea, a clade composed of
Assamioidea–Zalmoxoidea– Samooidea – Gonyleptoidea, is
well supported in virtually all analyses ( figure 1). Internal
resolution among these superfamilies had found conflict in
prior studies [19,27], as it probably required additional mol-
ecular data to resolve this rapid radiation of Laniatores
families. Phylogenomic data find the much-needed infor-
mation to resolve this clade, here assigned a Cretaceous age
(figure 2; electronic supplementary material, figure S3).
A sister group relationship of Dampetrus (an assamiid; the
only representative of Assamioidea included here) to
Zalmoxoidea– Samooidea is found with all data matrices
except V and VI, but does not receive support in most ana-
lyses with matrix I. Fewer genes seem to be necessary to
support fully a clade of Zalmoxoidea and Samooidea, which
was found in prior Sanger-based studies [27]. However,
Samooidea is paraphyletic with respect to Zalmoxoidea in
about half of the analyses (electronic supplementary material,
figure S1), although in others they are reciprocally monophy-
letic. The bona fide samooid Pellobunus from Panama is sister
group to the remaining members of this clade, followed by
a representative of Biantidae, Metabiantes from South Africa,
and by a clade of Zalmoxoidea, including an Amazonian
specimen we tentatively placed in the genus Escadabius
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
5
(Escadabiidae), and then the representatives of Fissiphalliidae
(Fissiphallius martensi) and Zalmoxidae (Zalmoxis,Pachylicus).
Except for the clade placing Metabiantes with the zalmoxoids,
relationships within this clade are stable (electronic sup-
plementary material, figure S2). Further samooid and
zalmoxoid missing families (Kimulidae, Stygnommatidae,
Guasiniidae and Icaleptidae; all exclusively Neotropical)
should be sampled to resolve the issue of the reciprocal
monophyly of the families.
Gonyleptoidea is restricted to an expanded Neotropics
(some gonyleptid species make it into Patagonia and
some cosmetids quite for north into the USA). Stygnopsidae
(Chapulobunus) is sometimes sister group to all other gonylep-
toids, followed by Agoristenidae (Avima), although the
position of Agoristenidae is not well resolved. Agoristenids
had been proposed as the sister group of the non-stygnopsid
gonyleptoids in previous analyses [27], as shown here in
some trees (figures 1– 3), but most matrices suggest a sister
group relationship of Agoristenidae and Stygnopsidae.
Stygnidae (Protimesius) is well supported as sister group to
all the remaining families, followed in a ladder-like fashion
by the families Cryptogeobiidae (Pseudopachylus), Cosmetidae
(Vonones), Gonyleptidae (Metagyndes), Manaosbiidae (Sarama-
cia) and Cranaidae (Phareicranaus). This topology is fully
compatible with more detailed recent analyses of Gonyleptoi-
dea [39,40,67], some of which consider Manaosbiidae and
Cranaidae subfamilies of Gonyleptidae, as originally proposed
by Roewer (see [67]), although this was not accepted in sub-
sequent studies [39,68]. We thus support a clade including
these three families, which has also been called Greater
Gonyleptidae (GG) [39,68].
(f ) Molecular dating
The molecular dating analyses for both calibration configur-
ations (i.e. the age of Eophalangium as the floor of Opiliones
or Cyphophthalmi) yielded very similar results, with
the main differences obtained between the autocorrelated
Ischyropsalididae
Triaenonychidae
Neopilionidae
Cranaidae
Stygnopsidae
Protolophidae
Sclerosomatidae
Zalmoxidae
Fissiphalliidae
Cosmetidae
Stygnidae
Synthetonychiidae
Assamiidae
Trogulidae
Acropsopilionidae
Cladonychiidae
Sironidae
Caddidae
Epedanidae
Samoidae
Neogoveidae
Phalangiidae
Nipponopsalididae
Escadabiidae
Cryptogeobiidae
Dicranolasmatidae
Taracidae
Agoristenidae
Sabaconidae
Phalangodidae
Manaosbiidae
Travuniidae
Podoctidae
Stylocellidae
Petrobunidae
Pettalidae
Sandokanidae
Gonyleptidae
Biantidae
Nippononychidae
CYPHOPHTHALMI
EUPNOI
DYSPNOI
LANIATORES
PHALANGIDA
N
e
o
op
ili
o
ni
n
da
a
a
e
e
Pr
ot
t
ol
op
hi
h
da
a
a
e
e
S
c
le
e
ro
so
ma
m
t
t
ti
da
d
e
e
P
h
al
l
ang
i
id
d
ae
e
e
Ca
d
dd
id
ae
CADDOIDEA
PHALANGIOIDEA
ACROPSOPILIONOIDEA
I
s
c
ch
y
ropsa
lidid
a
e
Ta
a
ra
c
id
a
e
Sa
a
b
ba
co
n
id
a
e
ISCHYROPSALIDOIDEA
TROGULOIDEA
SCOPULOPHTHALMI
STERNOPHTHALMI
TRIAENONYCHOIDEA
TRAVUNIOIDEA
SYNTHETONYCHOIDEA
PHALANGODOIDEA
SANDOKANOIDEA
EPEDANOIDEA
ASSAMIOIDEA
SAMOOIDEA
ZALMOXOIDEA
GONYLEPTOIDEA
PALPATORES
STYLOCELLOIDEA
SIRONOIDEA
Nemastomatidae
I
N
S
I
D
I
A
T
O
R
E
S
G
R
A
S
S
A
T
O
R
E
S
Figure 3. Summary tree of familial and superfamilial relationships of Opiliones supported in this study, with major nodes highlighted. (Online version in colour.)
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
6
and uncorrelated model analyses within each calibration con-
figuration (electronic supplementary material, figure S3). For
purposes of conservatism, we discuss results based on the
chronogram under the uncorrelated gamma multipliers
model using the age of Eophalangium as the minimum age
of Cyphophthalmi ( figure 2), but some of the divergence
dates may vary substantially.
Opiliones have oftenbeen used as examples of animals with
ancient and conservative biogeographic patterns, therefore suit-
able for vicariance biogeographic analyses [5,19]. One general
pattern observed here is a division between temperate Gond-
wana (the terranes that were once directly connected to
Antarctica) and the remaining landmasses, including, in some
cases, clades currently in tropical Gondwana. For example,
this is the case for Cyphophthalmi, with a main division
between the strictly temperate Gondwanan family Pettalidae
and the remaining families (this including Laurasian and tropi-
cal Gondwanan clades),or in Dyspnoi, with Acropsopilionidae,
being mostly distributed in temperate Gondwana, as the sister
group to the rest of the Dyspnoi families, restricted to the North-
ern Hemisphere. Within Eupnoi, Caddidae is mostly Laurasian,
but Phalangioidea once more divides into Neopilionidae,
restricted to temperate Gondwana (with the exception of
Thrasychiroides, which extends to the Atlantic rainforest [69]),
and the remaining families, mostly Laurasian, although some
secondarily extending southwards. Once more, Insidiatores,
although somehow unresolved, finds a division between the
predominantly temperate Gondwanan family Triaenonychidae
(or Triaenonychidae þSynthetonychiidae) and the Northern
Hemisphere Insidiatores (Travunioidea). In addition, Triaeno-
nychidae has a basal split between the Northern Hemisphere
Fumontana and the temperate Gondwanan clade (although
here it is represented bya single species), as shown in other pub-
lished phylogenies of Laniatores [27]. Laniatores depict several
other interesting patterns, including two clades of southeast
Asian families, Sandokanidae and Epedanoidea, while the
remaining species mostly appear to be of Tropical Gondwanan
origins, with some remarkable cases of range expansions
(e.g. trans-continental disjunctions in Assamiidae, Biantidae,
Podoctidae, Pyramidopidae and Zalmoxidae [27,60,70]).
Interestingly, the splits between temperate Gondwana
and the rest precede the breakup of Pangea (figure 2),
suggesting ancient regionalization across Pangea, as shown
in other groups of terrestrial invertebrates [71] and in the
early diversification of amphibians [72]. Splits between tropi-
cal Gondwana and Laurasia, both in Cyphophthalmi and in
Grassatores, seem to be much younger, and may be associ-
ated with the breakup of Pangea, possibly representing
true Gondwanan/Laurasian vicariant events, and not the
result of ancient cladogenesis and Pangean regionalization.
Detailed analyses with a much denser sampling within
each family should allow further scrutiny of these suggestive
distributions.
4. Conclusion
Our analysis of a large number of novel transcriptomes has
allowed us to propose a stable phylogeny of Opiliones
(figure 3). Such analyses of large data matrices have allowed
us to place all superfamilies of Opiliones (and 80% of the
families) in a resolved phylogenetic context, with only a
few spots to be sorted out in areas of the tree where sampling
was still limited. Our trees support most traditional relation-
ships within Opiliones and resolve some recalcitrant familial
relationships, such as a well-resolved Eupnoi phylogeny, the
rejection of Boreophthalmi, the monophyly of Insidiatores
and the placement of Stygnopsidae as the most basal family
of Gonyleptoidea, among others. We also show that Opi-
liones exhibit some splits reflecting ancestral Pangean
regionalization, whereas others conform with high fidelity
to the sequence of Pangean fragmentation, therefore consti-
tuting ideal model systems to understand ancient
biogeographic patterns.
Data accessibility. Electronic supplementarymaterial, figures S1– S3, tables
S1 and S2, and Extended material and methods are available from
https://dx.doi.org/10.6084/m9.figshare.c.3691975. All matrices are
available at the Dryad Digital Repository: http://dx.doi.org/10.
5061/dryad.fs8kv [73].
Authors’ contributions. R.F., P.P.S., A.L.T. and G.G. collected samples. R.F.
performed laboratory work and analyses. R.F., P.P.S. and G.G. wrote
the paper. All authors approved the final version of the manuscript.
Competing interests. We declare we have no competing interests.
Funding. The following funding sources were used: NSF grant DEB-
1457539 (G.G.); National Geographic grant no. 9043-11 (G.G.); Inter-
national Postdoctoral grant no. CNPq 200972/2013-8 (A.L.T.).
Fieldwork was supported by Putnam expedition grants from the
MCZ (G.G., R.F.); fieldwork to Reserva Ducke was supported by
CAPES/PVE no. AUX-PE-PVES 2510/2012 (A.L.T., G.G.); fieldwork
in the Philippines and Australia was supported by NSF DBI-1202751
(P.P.S.) and in the Philippines by a National Geographic grant to
Ronald M. Clouse and P.P.S.
Acknowledgements. Many colleagues assisted with fieldwork and
samples, and we are indebted to all of them for their contribution
of specimens and expertise, especially Jimmy Cabra, Ron Clouse,
Pı
´o Colmenares, Jesu
´s Alberto Cruz, O
´scar Francke, Guilherme
Gainett, Abel Pe
´rez Gonza
´lez, Gustavo Hormiga, Carlos Prieto,
Ricardo Pinto-da-Rocha, Cristiano Sampaio Porto, Willians Porto,
and Nobuo Tsurusaki. Ricardo Pinto-da-Rocha, editor Davide
Pisani. Two anonymous reviewers provided helpful comments that
improved this paper.
References
1. Dunlop JA, Anderson LI, Kerp H, Hass H. 2003
Preserved organs of Devonian harvestmen. Nature
425, 916. (doi:10.1038/425916a)
2. Dunlop JA. 2007 Paleontology. In Harvestmen: the
biology of Opiliones (eds R Pinto-da-Rocha, G
Machado, G Giribet), pp. 247–265. Cambridge, MA:
Harvard University Press.
3. Garwood RJ, Sharma PP, Dunlop JA, Giribet G. 2014
A new stem-group Palaeozoic harvestman revealed
through integration of phylogenetics and
development. Curr. Biol.24, 1– 7. (doi:10.1016/j.
cub.2014.03.039)
4. Boyer SL, Clouse RM, Benavides LR, Sharma P,
Schwendinger PJ, Karunarathna I, Giribet G. 2007
Biogeography of the world: a case study from
cyphophthalmid Opiliones, a globally distributed
group of arachnids. J. Biogeogr.34, 2070– 2085.
(doi:10.1111/j.1365-2699.2007.01755.x)
5. Giribet G, Kury AB. 2007 Phylogeny and
Biogeography. In Harvestmen: the biology of Opiliones
(eds R Pinto-da-Rocha, G Machado, G Giribet), pp.
62–87. Cambridge, MA: Harvard University Press.
6. Clouse RM, Giribet G. 2010 When Thailand was an
island—the phylogeny and biogeography of mite
harvestmen (Opiliones, Cyphophthalmi, Stylocellidae)
in Southeast Asia. J. Biogeogr.37, 1114– 1130.
(doi:10.1111/j.1365-2699.2010.02274.x)
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
7
7. Giribet G, Sharma PP. 2015 Evolutionary biology of
harvestmen (Arachnida, Opiliones). Annu. Rev.
Entomol.60, 157– 175. (doi:10.1146/annurev-ento-
010814-021028)
8. Giribet G et al. 2012 Evolutionary and
biogeographical history of an ancient and global
group of arachnids (Arachnida: Opiliones:
Cyphophthalmi) with a new taxonomic
arrangement. Biol. J. Linn. Soc.105, 92–130.
(doi:10.1111/j.1095-8312.2011.01774.x)
9. Machado G. 2007 Maternal or paternal egg
guarding? Revisiting parental care in triaenonychid
harvestmen (Opiliones). J. Arachnol.35, 202–204.
(doi:10.1636/SH06-14.1)
10. Machado G, Macı
´as-Ordo
´n
˜ez R. 2007 Reproduction.
In Harvestmen: the biology of Opiliones (eds R Pinto-
da-Rocha, G Machado, G Giribet), pp. 414– 454.
Cambridge, MA: Harvard University Press.
11. Requena GS, Buzatto BA, Martins EG, Machado G.
2012 Paternal care decreases foraging activity
and body condition, but does not impose
survival costs to caring males in a Neotropical
arachnid. PLoS ONE 7, e46701. (doi:10.1371/journal.
pone.0046701)
12. Buzatto BA, Tomkins JL, Simmons LW, Machado G.
2014 Correlated evolution of sexual dimorphism and
male dimorphism in a clade of neotropical
harvestmen. Evolution 68, 1671– 1686. (doi:10.
1111/evo.12395)
13. Martens J. 1976 Genitalmorphologie, System und
Phylogenie der Weberknechte (Arachnida,
Opiliones). Entomol. Germ.3, 51–68.
14. Martens J. 1980 Versuch eines Phylogenetischen
Systems der Opiliones. In Proc. 8th International
Congress of Arachnology, pp. 355– 360. Vienna,
Austria: Verlag H. Egerman.
15. Martens J, Hoheisel U, Go
¨tze M. 1981 Vergleichende
Anatomie der Legero
¨hren der Opiliones als Beitrag
zur Phylogenie der Ordnung (Arachnida). Zool. Jb.
Anat.105, 13– 76.
16. Shultz JW. 1998 Phylogeny of Opiliones (Arachnida):
an assessment of the ‘Cyphopalpatores’ concept.
J. Arachnol.26, 257– 272.
17. Giribet G, Dunlop JA. 2005 First identifiable
Mesozoic harvestman (Opiliones: Dyspnoi) from
Cretaceous Burmese amber. Proc. R. Soc. B 272,
1007–1013. (doi:10.1098/rspb.2005.3063)
18. Shultz JW, Regier JC. 2001 Phylogenetic analysis of
Phalangida (Arachnida, Opiliones) using two nuclear
protein-encoding genes supports monophyly of
Palpatores. J. Arachnol.29, 189–200. (doi:10.1636/
0161-8202(2001)029[0189:PAOPAO]2.0.CO;2)
19. Giribet G, Vogt L, Pe
´rez Gonza
´lez A, Sharma P, Kury
AB. 2010 A multilocus approach to harvestman
(Arachnida: Opiliones) phylogeny with emphasis on
biogeography and the systematics of Laniatores.
Cladistics 26, 408– 437. (doi:10.1111/j.1096-0031.
2009.00296.x)
20. Groh S, Giribet G. 2015 Polyphyly of Caddoidea,
reinstatement of the family Acropsopilionidae in
Dyspnoi, and a revised classification system of
Palpatores (Arachnida, Opiliones). Cladistics 31,
277–290. (doi:10.1111/cla.12087)
21. Hedin M, Derkarabetian S, McCormack M, Richart C,
Shultz JW. 2010 The phylogenetic utility of the
nuclear protein-coding gene EF-1 alpha for
resolving recent divergences in Opiliones,
emphasizing intron evolution. J. Arachnol.38,
9– 20. (doi:10.1636/HA09-49.1)
22. Giribet G, Rambla M, Carranza S, Bagun
˜a
`J, Riutort
M, Ribera C. 1999 Phylogeny of the arachnid order
Opiliones (Arthropoda) inferred from a combined
approach of complete 18S and partial 28S ribosomal
DNA sequences and morphology. Mol. Phylogenet.
Evol.11, 296– 307. (doi:10.1006/mpev.1998.0583)
23. Giribet G, Edgecombe GD, Wheeler WC, Babbitt C.
2002 Phylogeny and systematic position of
Opiliones: a combined analysis of chelicerate
relationships using morphological and molecular
data. Cladistics 18, 5– 70. (doi:10.1006/clad.
2001.0185)
24. Hedin M, Starrett J, Akhter S, Scho
¨nhofer AL, Shultz
JW. 2012 Phylogenomic resolution of Paleozoic
divergences in harvestmen (Arachnida, Opiliones)
via analysis of next-generation transcriptome data.
PLoS ONE 7, e428888. (doi:10.1371/journal.pone.
0042888.g001)
25. Sharma PP, Giribet G. 2014 A revised dated
phylogeny of the arachnid order Opiliones. Front.
Genet.5, 255. (doi:10.3389/fgene.2014.00255)
26. Sharma PP, Kaluziak S, Pe
´rez-Porro AR, Gonza
´lez VL,
Hormiga G, Wheeler WC, Giribet G. 2014
Phylogenomic interrogation of Arachnida reveals
systemic conflicts in phylogenetic signal. Mol.
Biol. Evol.31, 2963–2984. (doi:10.1093/molbev/
msu235)
27. Sharma PP, Giribet G. 2011 The evolutionary and
biogeographic history of the armoured
harvestmen—Laniatores phylogeny based on ten
molecular markers, with the description of two new
families of Opiliones (Arachnida). Invertebr. Syst.25,
106–142. (doi:10.1071/IS11002)
28. Richart CH, Hedin M. 2013 Three new species in the
harvestmen genus Acuclavella (Opiliones, Dyspnoi,
Ischyropsalidoidea), including description of male
Acuclavella quattuor Shear, 1986. ZooKeys 311,
19–68. (doi:10.3897/Zookeys.311.2920)
29. Scho
¨nhofer AL, McCormack M, Tsurusaki N, Martens
J, Hedin M. 2013 Molecular phylogeny of the
harvestmen genus Sabacon (Arachnida: Opiliones:
Dyspnoi) reveals multiple Eocene– Oligocene
intercontinental dispersal events in the Holarctic.
Mol. Phylogenet. Evol.66, 303– 315. (doi:10.1016/j.
ympev.2012.10.001)
30. Shear WA, Gruber J. 1983 The opilionid subfamily
Ortholasmatinae (Opiliones, Troguloidea,
Nemastomatidae). Am. Mus. Novit.2757, 1– 65.
31. Shear WA. 1986 A cladistic analysis of the opilionid
superfamily Ischyropsalidoidea, with descriptions of
the new family Ceratolasmatidae, the new genus
Acuclavella, and four new species. Am. Mus. Novit.
2844, 1– 29.
32. Shear WA. 1980 A review of the Cyphophthalmi of
the United States and Mexico, with a proposed
reclassification of the suborder (Arachnida,
Opiliones). Am. Mus. Novit.2705, 1– 34.
33. Hunt GS, Cokendolpher JC. 1991 Ballarrinae, a new
subfamily of harvestmen from the Southern
Hemisphere (Arachnida, Opiliones, Neopilionidae).
Rec. Aust. Mus.43, 131– 169. (doi:10.3853/j.0067-
1975.43.1991.45)
34. Scho
¨nhofer AL. 2013 A taxonomic catalogue of the
Dyspnoi Hansen and Sørensen, 1904 (Arachnida:
Opiliones). Zootaxa 3679, 1–68. (doi:10.11646/
zootaxa.3679.1.1)
35. Hedin M, Tsurusaki N, Macı
´as-Ordo
´n
˜ez R, Shultz JW.
2012 Molecular systematics of sclerosomatid
harvestmen (Opiliones, Phalangioidea,
Sclerosomatidae): geography is better than taxonomy
in predicting phylogeny. Mol. Phylogenet. Evol.62,
224 – 236. (doi:10.1016/j.ympev.2011.09.017)
36. Ve
´lez S, Ferna
´ndez R, Giribet G. 2014 A molecular
phylogenetic approach to the New Zealand species
of Enantiobuninae (Opiliones: Eupnoi:
Neopilionidae). Invertebr. Syst.28, 565– 589.
(doi:10.1071/IS14030)
37. Richart CH, Hayashi CY, Hedin M. 2016
Phylogenomic analyses resolve an ancient
trichotomy at the base of Ischyropsalidoidea
(Arachnida, Opiliones) despite high levels of gene
tree conflict and unequal minority resolution
frequencies. Mol. Phylogenet. Evol.95, 171– 182.
(doi:10.1016/j.ympev.2015.11.010)
38. Sharma PP, Prieto CE, Giribet G. 2011 A new family
of Laniatores (Arachnida: Opiliones) from the
Afrotropics. Invertebr. Syst.25, 143– 154. (doi:10.
1071/IS11003)
39. Kury AB. 2014 Why does the Tricommatinae position
bounce so much within Laniatores? A cladistic
analysis, with description of a new family of
Gonyleptoidea (Opiliones, Laniatores). Zool. J. Linn.
Soc.172, 1– 48. (doi:10.1111/zoj.12165)
40. Bragagnolo C, Hara MR, Pinto-da-Rocha R. 2015 A
new family of Gonyleptoidea from South America
(Opiliones, Laniatores). Zool. J. Linn. Soc.173,
296–319. (doi:10.1111/zoj.12207)
41. Ferna
´ndez R, Hormiga G, Giribet G. 2014
Phylogenomic analysis of spiders reveals
nonmonophyly of orb weavers. Curr. Biol.24,
1772–1777. (doi:10.1016/j.cub.2014.06.035)
42. Ferna
´ndez R, Giribet G. 2015 Unnoticed in the
tropics: phylogenomic resolution of the poorly
known arachnid order Ricinulei (Arachnida). R. Soc.
Open Sci.2, 150065. (doi:10.1098/rsos.150065)
43. Sharma PP, Ferna
´ndez R, Esposito LA, Gonza
´lez-
Santilla
´n E, Monod L. 2015 Phylogenomic resolution
of scorpions reveals multilevel discordance with
morphological phylogenetic signal. Proc. R. Soc. B
282, 20142953. (doi:10.1098/rspb.2014.2953)
44. Bond JE, Garrison NL, Hamilton CA, Godwin RL,
Hedin M, Agnarsson I. 2014 Phylogenomics resolves
a spider backbone phylogeny and rejects a prevailing
paradigm for orb web evolution. Curr. Biol.24,
1765– 1771. (doi:10.1016/j.cub.2014.06.034)
45. Ferna
´ndez R, Edgecombe GD, Giribet G. 2016
Exploring phylogenetic relationships within
Myriapoda and the effects of matrix composition
and occupancy on phylogenomic reconstruction. Syst.
Biol.65, 871– 889. (doi:10.1093/sysbio/syw041)
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
8
46. Misof B et al. 2014 Phylogenomics resolves the
timing and pattern of insect evolution. Science 346,
763–767. (doi:10.1126/science.1257570)
47. Altenhoff AM, Gil M, Gonnet GH, Dessimoz C. 2013
Inferring hierarchical orthologous groups from
orthologous gene pairs. PLoS ONE 8, e53786.
(doi:10.1371/journal.pone.0053786)
48. Ferna
´ndez R, Laumer CE, Vahtera V, Libro S, Kaluziak
S, Sharma PP, Pe
´rez-Porro AR, Edgecombe GD,
Giribet G. 2014 Evaluating topological conflict in
centipede phylogeny using transcriptomic data sets.
Mol. Biol. Evol.31, 1500– 1513. (doi:10.1093/
molbev/msu108)
49. Zoller S, Schneider A. 2013 Improving phylogenetic
inference with a semiempirical amino acid
substitution model. Mol. Biol. Evol.30, 469– 479.
(doi:10.1093/molbev/mss229)
50. Aberer AJ, Stamatakis A. 2013 ExaML: exascale
maximum likelihood: program and documentation.
See http://sco.h-its.org/exelixis/web/software/
examl/index.html.
51. Aberer AJ, Kobert K, Stamatakis A. 2014 ExaBayes:
massively parallel Bayesian tree inference for the
whole-genome era. Mol. Biol. Evol.31, 2553– 2556.
(doi:10.1093/molbev/msu236)
52. Lartillot N, Rodrigue N, Stubbs D, Richer J. 2013
PhyloBayes MPI: phylogenetic reconstruction with
infinite mixtures of profiles in a parallel
environment. Syst. Biol.62, 611– 615. (doi:10.1093/
Sysbio/Syt022)
53. Lartillot N, Philippe H. 2004 A Bayesian
mixture model for across-site heterogeneities
in the amino-acid replacement process. Mol. Biol.
Evol.21, 1095– 1109. (doi:10.1093/molbev/
msh112)
54. Ku¨ck P, Struck TH. 2014 BaCoCa—a heuristic
software tool for the parallel assessment of
sequence biases in hundreds of gene and taxon
partitions. Mol. Phylogenet. Evol.70, 94– 98.
(doi:10.1016/j.ympev.2013.09.011)
55. Yang ZH, Rannala B. 2006 Bayesian estimation of
species divergence times under a molecular clock
using multiple fossil calibrations with soft
bounds. Mol. Biol. Evol.23, 212–226. (doi:10.1093/
molbev/msj024)
56. Benavides LR, Giribet G. 2013 A revision of selected
clades of Neotropical mite harvestmen (Arachnida,
Opiliones, Cyphophthalmi, Neogoveidae) with the
description of eight new species. Bull. Mus. Comp.
Zool.161, 1 – 44. (doi:10.3099/0027-4100-161.1.1)
57. Kury AB. 2013 Order Opiliones Sundevall, 1833.
Zootaxa 3703, 27–33. (doi:10.11646/zootaxa.3703.
1.7)
58. Shear WA. 2010 New species and records of
ortholasmatine harvestmen from Me
´xico, Honduras,
and the western United States (Opiliones,
Nemastomatidae, Ortholasmatinae). ZooKeys 52,
9– 45. (doi:10.3897/zookeys.52.471)
59. Dunlop JA, Selden PA, Giribet G. 2016 Penis
morphology in a Burmese amber harvestman. Sci.
Nat.103, 11. (doi:10.1007/s00114-016-1337-4)
60. Cruz-Lo
´pez JA, Proud DN, Pe
´rez-Gonza
´lez A. 2016
When troglomorphism dupes taxonomists:
morphology and molecules reveal the first
pyramidopid harvestman (Arachnida, Opiliones,
Pyramidopidae) from the New World. Zool. J. Linn.
Soc.177, 602– 620. (doi:10.1111/zoj.12382)
61. Sharma PP et al. 2017 A multilocus phylogeny of
Podoctidae (Arachnida, Opiliones, Laniatores) and
parametric shape analysis reveal the disutility of
subfamilial nomenclature in armored harvestman
systematics. Mol. Phylogenet. Evol.106, 164– 173.
(doi:10.1016/j.ympev.2016.09.019)
62. Kury AB. 1993 Ana
´lise filogene
´tica de
Gonyleptoidea (Arachnida, Opiliones, Laniatores).
Thesis, Universidade de Sa
˜o Paulo, Sa
˜o Paulo, Brazil.
63. Kury AB. 2015 Opiliones are no longer the same-on
suprafamilial groups in harvestmen (Arthropoda:
Arachnida). Zootaxa 3925, 301– 340. (doi:10.
11646/zootaxa.3925.3.1)
64. Forster RR. 1954 The New Zealand harvestmen
(sub-order Laniatores). Canterbury Mus. Bull.2,
1–329.
65. Sharma P, Giribet G. 2009 Sandokanid phylogeny
based on eight molecular markers—the
evolution of a southeast Asian endemic family of
Laniatores (Arachnida, Opiliones). Mol. Phylogenet.
Evol.52, 432– 447. (doi:10.1016/j.ympev.2009.
03.013)
66. Selden PA, Dunlop JA, Giribet G, Zhang W, Ren D.
2016 The oldest armoured harvestman (Arachnida:
Opiliones: Laniatores), from Upper Cretaceous
Myanmar amber. Cretaceous Res.65, 206– 212.
(doi:10.1016/j.cretres.2016.05.004)
67. Pinto-da-Rocha R, Bragagnolo C, Marques FPL,
Antunes Junior M. 2014 Phylogeny of harvestmen
family Gonyleptidae inferred from a multilocus
approach (Arachnida: Opiliones). Cladistics 30,
519–539. (doi:10.1111/cla.12065)
68. Kury AB, Villarreal MO. 2015 The prickly blade
mapped: establishing homologies and a
chaetotaxy for macrosetae of penis ventral plate in
Gonyleptoidea (Arachnida, Opiliones, Laniatores).
Zool. J. Linn. Soc.174, 1–46. (doi:10.1111/
zoj.12225)
69. Pinto-da-Rocha R, Bragagnolo C, Tourinho AL.
2014 Three new species of Thrasychiroides Soares &
Soares, 1947 from Brazilian Mountains (Opiliones,
Eupnoi, Neopilionidae). Zootaxa 3869, 469– 482.
(doi:10.11646/zootaxa.3869.4.9)
70. Sharma PP, Giribet G. 2012 Out of the
Neotropics: late Cretaceous colonization
of Australasia by American arthropods. Proc. R.
Soc. B 279, 3501– 3509. (doi:10.1098/rspb.2012.
0675)
71. Murienne J, Daniels SR, Buckley TR, Mayer G, Giribet
G. 2014 A living fossil tale of Pangaean
biogeography. Proc. R. Soc. B 281, 20132648.
(doi:10.1098/rspb.2013.2648)
72. San Mauro D, Vences M, Alcobendas M, Zardoya
R, Meyer A. 2005 Initial diversification of
living amphibians predated the breakup of
Pangaea. Am. Nat.165, 590 – 599. (doi:10.1086/
429523)
73. Ferna
´ndez R, Sharma PP, Tourinho AL, Giribet G.
2017 The Opiliones tree of life: shedding light on
harvestmen relationships through transcriptomics.
Dryad Digital Repository. (http://dx.doi.org/10.5061/
dryad.fs8kv)
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20162340
9
