Was There a Cambrian Explosion on Land? The Case of Arthropod Terrestrialization

Abstract

Arthropods, the most diverse form of macroscopic life in the history of the Earth, originated in the sea. Since the early Cambrian, at least ~518 million years ago, these animals have dominated the oceans of the world. By the Silurian–Devonian, the fossil record attests to arthropods becoming the first animals to colonize land, However, a growing body of molecular dating and palaeontological evidence suggests that the three major terrestrial arthropod groups (myriapods, hexapods, and arachnids), as well as vascular plants, may have invaded land as early as the Cambrian–Ordovician. These dates precede the oldest fossil evidence of those groups and suggest an unrecorded continental “Cambrian explosion” a hundred million years prior to the formation of early complex terrestrial ecosystems in the Silurian–Devonian. We review the palaeontological, phylogenomic, and molecular clock evidence pertaining to the proposed Cambrian terrestrialization of the arthropods. We argue that despite the challenges posed by incomplete preservation and the scarcity of early Palaeozoic terrestrial deposits, the discrepancy between molecular clock estimates and the fossil record is narrower than is often claimed. We discuss strategies for closing the gap between molecular clock estimates and fossil data in the evolution of early ecosystems on land.

Full text

Citation: Tihelka, E.; Howard, R.J.;
Cai, C.; Lozano-Fernandez, J. Was
There a Cambrian Explosion on
Land? The Case of Arthropod
Terrestrialization. Biology 2022,11,
1516. https://doi.org/10.3390/
biology11101516
Academic Editors: Mary
H. Schweitzer and Ferhat Kaya
Received: 5 September 2022
Accepted: 14 October 2022
Published: 17 October 2022
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biology
Review
Was There a Cambrian Explosion on Land? The Case of
Arthropod Terrestrialization
Erik Tihelka 1, Richard J. Howard 2, Chenyang Cai 1,3 and Jesus Lozano-Fernandez 1,4 ,*
1School of Earth and Biological Sciences, University of Bristol, Bristol BS8 1TQ, UK
2Department of Earth Sciences, The Natural History Museum, London SW7 5BD, UK
3
State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, and
Center for Excellence in Life and Paleoenvironment, Chinese Academy of Sciences, Nanjing 210008, China
4Department of Genetics, Microbiology and Statistics & Biodiversity Research Institute (IRBio),
University of Barcelona, 08028 Barcelona, Spain
*Correspondence: jesus.lozano@ub.edu
Simple Summary:
The transition of life from the aquatic realm onto land represented one of the
fundamental episodes in the evolution of the Earth that laid down the foundations for modern
ecosystems as we know them today. This key transition in the history of life is poorly known, owing
to the scarcity of ancient terrestrial fossil deposits; complex terrestrial ecosystems with plants and
animals appear in the fossil record during the Silurian and Devonian. However, recent molecular
clock studies and new lines of palaeontological evidence point to a possibly much earlier origin
of life on land, dating back as far as the Cambrian. Here, we review this controversy, using the
arthropods as a case study of the possible cryptic Cambrian explosion on land. In particular, we
highlight approaches for reconciling the disagreement between molecular clock estimates and the
fossil record for the arthropod colonization of land.
Abstract:
Arthropods, the most diverse form of macroscopic life in the history of the Earth, originated
in the sea. Since the early Cambrian, at least ~518 million years ago, these animals have dominated
the oceans of the world. By the Silurian–Devonian, the fossil record attests to arthropods becoming
the first animals to colonize land, However, a growing body of molecular dating and palaeontological
evidence suggests that the three major terrestrial arthropod groups (myriapods, hexapods, and
arachnids), as well as vascular plants, may have invaded land as early as the Cambrian–Ordovician.
These dates precede the oldest fossil evidence of those groups and suggest an unrecorded continental
“Cambrian explosion” a hundred million years prior to the formation of early complex terrestrial
ecosystems in the Silurian–Devonian. We review the palaeontological, phylogenomic, and molecular
clock evidence pertaining to the proposed Cambrian terrestrialization of the arthropods. We argue
that despite the challenges posed by incomplete preservation and the scarcity of early Palaeozoic
terrestrial deposits, the discrepancy between molecular clock estimates and the fossil record is
narrower than is often claimed. We discuss strategies for closing the gap between molecular clock
estimates and fossil data in the evolution of early ecosystems on land
Keywords:
terrestrialization; artrhopods; Cambrian explosion; molecular clocks; palaeontology;
phylogenomics
1. Introduction
Molecular clocks estimate that life on Earth originated over 4 billion of years ago
(Ga), perhaps shortly after the formation of our planet [
1
], with direct evidence provided
by the remains of putative unicellular organisms at around 3.5 Ga (e.g., [
2
–
4
]). However,
the emergence of complex multicellular organisms, such as animals, plants and fungi,
only occurred during the last 1000 million years [
5
] (but see [
6
] for older estimates). The
Biology 2022,11, 1516. https://doi.org/10.3390/biology11101516 https://www.mdpi.com/journal/biology

Biology 2022,11, 1516 2 of 18
origin of animals gave rise to an enormous diversity of multicellular body plans, all with
a complex embryonic development. This diversity of body plans is already seen in the
exceptional early fossil record of animals, during the “Cambrian explosion”, beginning
around 540 million years ago (Ma) and concluding perhaps as quickly as 521 Ma [
7
].
During this interval, most major animal phyla appeared almost simultaneously, from
a geological perspective, and persisted throughout the Phanerozoic [
8
,
9
]). The often
unfamiliar body plans of Cambrian marine animals have been preserved on a number
of sites with exceptional preservation, known as the Burgess Shale-type (BST) Konservat-
Lagerstätten, which provide a unique snapshot of the soft-bodied Cambrian biota in the
sea [
10
]. A diverse and abundant marine arthropod fauna is evidenced by the fossil record
from at least ~518 Ma, corresponding to the minimum age of the Chengjiang Biota of
Yunnan Province, southwestern China; the oldest reliably dated BST [11].
Animals, plants, and life in general, have marine origins [
12
]. Only a handful of animal
phyla contain lineages that can complete each phase of their life cycle outside of moisture-
rich environments and can therefore be considered fully terrestrial. This is because land
represents a new and hostile environment for marine organisms, with obstacles to overcome
ranging from respiration, reproduction, feeding style, and mechanical support [
13
]. Among
these, the most well-known examples are in vertebrates (reptiles, birds and mammals) and
of course arthropods, invertebrates with jointed legs and exoskeletons such as spiders and
insects. Additionally, soft-bodied groups with generally poor fossil records [
14
,
15
], such as
molluscs (including the land snails and slugs [
16
]), onychophorans (velvet worms [
17
]),
annelids (including earthworms [
18
]), nematoids (roundworms and horsehair worms,
including many parasitic groups that have followed their hosts on land [
18
–
21
]), tardigrades
(water bears [
22
]), and platyhelminthes (flatworms [
23
]) contain land-living lineages, but
these are mostly dependent on moisture-rich terrestrial environments for survival. Life on
land requires a series of adaptations that may be paralleled across different groups—we
can refer to this as terrestrialization: the process by which aquatic organisms adapt to
terrestrial life. Terrestrialization is a fascinating field of study in evolutionary biology.
Much literature has addressed terrestrialization at the physiological level in arthropods
(see review in [
24
,
25
]). However, most studies have been conducted on isolated lineages
and have not taken full advantage of the comparative approach between diverse terrestrial
groups [
26
]. Multiple and independent terrestrialization events allow comparisons of
alternative solutions taken up by different groups to the same adaptive challenge, and
represent a powerful tool to understand adaptation in an evolutionary framework. This
information is, at the same time, necessary to be able to carry out comparative analyses
and estimate the timing and rate of emergence of terrestrial adaptations. Although animal
phylogenetic diversity (understood as the diversity of body plans) may be higher in the
marine realm, terrestrial biodiversity is clearly higher in terms of the number of species—
particularly due to the unparalleled species richness of insects [
27
]. Understanding animal
terrestrialization is thus crucial to understanding the origins of biodiversity on Earth and
the mechanisms underpinning evolutionary adaptation [28].
There is fossil evidence of simple terrestrial ecosystems formed by single-cell or-
ganisms dating back 1000 Ma [
29
]). The earliest complex terrestrial ecosystems record a
fascinating transition in the history of life. Before the Palaeozoic, the only terrestrial life was
unicellular, which, until recently, could only be deduced from indirect evidence [
30
]. It was
during the Palaeozoic that plants and animals began to colonize the Earth’s landmasess [
31
],
with plants appearing in the fossil record in the form of microfossils called cryptospores
in the Middle Ordovician, around 470 Ma, with potential vascular land plants appearing
shortly at ~458 Ma [
32
]. In the case of arthropods, with certain terrestrial myriapods and
arachnids from the Silurian–Devonian [
33
,
34
]. Hence, the conventional view of the evolu-
tion of terrestrial ecosystems posits that during the Silurian–Devonian, animals and plants
diversified on land, which was presumably void of complex organisms, bathed in lethal
UV rays, and with low atmospheric oxygen (e.g., [
35
,
36
]). This model has however recently
been challenged by molecular clock dating studies [
25
,
37
,
38
] and new discoveries of Palaeo-

Biology 2022,11, 1516 3 of 18
zoic stem groups of terrestrial lineage [
39
,
40
], which imply a substantially earlier, Cambrian
to Ordovician, origin of complex terrestrial ecosystems, comparable to a “Cambrian explo-
sion on land”. Secondly, updated reconstructions of Devonian-Carboniferous atmospheric
oxygen suggest that this period did not suffer from substantially low atmospheric oxygen
as stipulated earlier [
41
,
42
]. Meanwhile, terrestrial sedimentary rock units older than the
Early Devonian are rare worldwide (e.g., [
43
–
47
]). For example, Western Europe, one of the
best explored regions of the world from a palaeontological point of view, has virtually no
terrestrial sedimentary rock outcrops older than the latest Silurian [
15
,
48
,
49
]. The scarcity
of preserved rock units imposes an important constraint on the preservation potential
of the earliest terrestrial ecosystems. It has been argued that the scarcity of terrestrial
organisms from this period may be due to limited surviving fossiliferous sediments rather
than because they did not exist in the first place [
42
,
50
]. This emerging paradigm may
imply up to 100 million years of discordance between when diverse terrestrial ecosystems
become represented in the fossil record and their putative origin.
In this brief review, we introduce the timescale of arthropod terrestrialization. Arthro-
pods are represented among the oldest fossil records of animals (Figure 1), and represent
the bulk of animal diversity on land today, with more than a million described species [
51
].
The oldest arthropod fossils are undoubtedly marine. They include the trilobites, with
representatives dating back to the early Cambrian, ~521 Ma [
52
], and trace fossils indicating
the presence of arthropod locomotion from at least ~528 Ma [
53
]. In arthropods, there have
been a minimum of three to four major terrestrial invasions during the Palaeozoic: that of
hexapods (which includes insects and kin), isopods (a group of crustaceans), myriapods,
and that of arachnids—assuming that the latter forms a monophyletic group. The multiple
and independent terrestrializations in arthropods provide a unique macroevolutionary
case study into adaptative solutions embraced by different groups in response to the same
challenge. More broadly, the topic of animal and plant terrestrialization provides an excit-
ing opportunity to study a crucial ecosystem-wide transition that shaped the world we find
so familiar today, during an elusive epoch of Earth’s history that left little direct physical
evidence. However, to carry out these studies it is necessary to: (i) clarify how many
land settlements have occurred independently in different arthropod lineages, (ii) estimate
when these terrestrialization processes occurred and how long they lasted, and (iii) estab-
lish robustly which is the aquatic sister group of each terrestrial lineage. We provide an
overview of recent progress in these questions and evaluate the support for the argument
of a Cambrian explosion on land.
Biology 2022, 11, x 4 of 19
Figure 1. Fossil evidence of arthropod terrestrialization. (A) Traces and the body fossil of the horse-
shoe crab that made it, Mesolimulus walchi, morphologically resembling modern forms; (B) recon-
struction of a terrestrial Cambrian ichnofossil, possibly made by the euthycarcinoid Mosineia, a
group in kinship with myriapods; (C) Section through the abdomen of a trigonotarbid arachnid
preserved in the Early Devonian Rhynie chert, revealing book lungs (bl), a possible trace of the gut
(gu?), and sections through the legs (lg); (D) Carbonised body fossil of a trigonotarbid arachnid
Palaeotarbus jerami from the Silurian Ludford Lane; (E) Putative myriapod mandibles from the Silu-
rian Ludford Lane; (F) Millipede Pneumodesmus newmani from the Lower Devonian of Cowie Har-
bour (Scotland), presenting spiracles (sp) and legs (lg); (G) Eurypterid Eurypterus remipes from the
Silurian; (H) Palaeoreconstruction of the Devonian scorpion Waeringoscorpio westerwaldensis, with
filamentous gills that suggest a potential aquatic adaptation. Image sources: Wikimedia Commons
Illustration authors: (B) Haug; (C–E), Erik Tihelka; (H) Junnn11 (@ni075). Institutional repositories:
(C–F) National Museum of Scotland, Edinburgh: R.08.14 & G.2001.109.1; (D,E) Ulster Museum, Bel-
fast: K25850 & LL1.6-23; (G) Generaldirektion Kulturelles Erbe, Direktion Archiolo-
gie/Erdgeschichte, Mainz, Germany, based on PWL2007/5000-LS. Scale bars: (C,D) 500 μm, (E) 250
μm, (G) ~10 mm.
2. Origin and Terrestrialization of Arthropods
2.1. Arthropod Origins
It is difficult to precisely estimate terrestrial arthropod biodiversity in deep time due
to the caveats of the fossil record; terrestrial arthropod fossils are usually limited to sites
of exceptional preservation known as Konservat-Lagerstätten, and therefore their strati-
graphic and environmental distribution is discontinuous. However, we can suppose that,
as in the modern biosphere, arthropods were probably the largest component of the di-
versity and abundance of Palaeozoic land animals, given the lack of initial competition
and the phylogenetic diversity of those that are present in the terrestrial Palaeozoic fossil
record. Indeed, arthropods are likely to have been the dominant animal group in terms of
biodiversity in perpetuity for the past 520 million years [54]. Arthropods are characterised
by presenting internal and external body segmentation with regional specialisations (tag-
mosis: in the case of insects, for example, they possess a thorax where legs and wings are
inserted while there are no extremities in the abdomen); an external skeleton composed
of articulated sclerotized parts; body segments that originally had associated articulated
Figure 1.
Fossil evidence of arthropod terrestrialization. (
A
) Traces and the body fossil of the horseshoe
crab that made it, Mesolimulus walchi, morphologically resembling modern forms; (B) reconstruction

Biology 2022,11, 1516 4 of 18
of a terrestrial Cambrian ichnofossil, possibly made by the euthycarcinoid Mosineia, a group in
kinship with myriapods; (
C
) Section through the abdomen of a trigonotarbid arachnid preserved
in the Early Devonian Rhynie chert, revealing book lungs (bl), a possible trace of the gut (gu?), and
sections through the legs (lg); (
D
) Carbonised body fossil of a trigonotarbid arachnid Palaeotarbus
jerami from the Silurian Ludford Lane; (
E
) Putative myriapod mandibles from the Silurian Ludford
Lane; (
F
) Millipede Pneumodesmus newmani from the Lower Devonian of Cowie Harbour (Scotland),
presenting spiracles (sp) and legs (lg); (
G
) Eurypterid Eurypterus remipes from the Silurian; (
H
) Palae-
oreconstruction of the Devonian scorpion Waeringoscorpio westerwaldensis, with filamentous gills that
suggest a potential aquatic adaptation. Image sources: Wikimedia Commons Illustration authors: (
B
)
Haug; (
C
–
E
), Erik Tihelka; (
H
) Junnn11 (@ni075). Institutional repositories: (
C
–
F
) National Museum
of Scotland, Edinburgh: R.08.14 & G.2001.109.1; (
D
,
E
) Ulster Museum, Belfast: K25850 & LL1.6-23;
(
G
) Generaldirektion Kulturelles Erbe, Direktion Archiologie/Erdgeschichte, Mainz, Germany, based
on PWL2007/5000-LS. Scale bars: (C,D) 500 µm, (E) 250 µm, (G) ~10 mm.
2. Origin and Terrestrialization of Arthropods
2.1. Arthropod Origins
It is difficult to precisely estimate terrestrial arthropod biodiversity in deep time due
to the caveats of the fossil record; terrestrial arthropod fossils are usually limited to sites of
exceptional preservation known as Konservat-Lagerstätten, and therefore their stratigraphic
and environmental distribution is discontinuous. However, we can suppose that, as in
the modern biosphere, arthropods were probably the largest component of the diversity
and abundance of Palaeozoic land animals, given the lack of initial competition and the
phylogenetic diversity of those that are present in the terrestrial Palaeozoic fossil record.
Indeed, arthropods are likely to have been the dominant animal group in terms of biodi-
versity in perpetuity for the past 520 million years [
54
]. Arthropods are characterised by
presenting internal and external body segmentation with regional specialisations (tagmo-
sis: in the case of insects, for example, they possess a thorax where legs and wings are
inserted while there are no extremities in the abdomen); an external skeleton composed
of articulated sclerotized parts; body segments that originally had associated articulated
limbs; growth through successive moults (ecdysis); and an open circulatory system with
a dorsal heart with lateral valves [
55
]. This set of unique characteristics suggests that
they are a monophyletic group (descendants of a common ancestor who possessed the
diagnostic characteristics of the lineage). Arthropods are represented by chelicerates (with
arachnids such as spiders and scorpions, and marine groups such as pycnogonids and
horseshoe crabs); myriapods (such as millipedes and centipedes); hexapods (containing
insects) and predominantly aquatic ‘crustaceans’ (for example crabs and prawns), which
are collectively known as pancrustaceans; and include important extinct groups, such as the
trilobites (Figure 2). Their abundance makes arthropods ecologically essential; for example,
myriapods are important processors of detritus in forests, and termites consume such large
amounts of cellulose that they are significant for the carbon cycle and atmospheric gas
composition [
56
]. Without arthropods, life and ecosystems on Earth would be radically
different. Their surprising diversity (which exceeds 75% of all living species described [
57
])
can help to elucidate the patterns and processes of macroevolution.
The earliest animals we know as land-dwelling were arthropods [
58
]. Evaluating the
earliest fossil evidence of arthropod life on land can rely on two approaches—phylogenetic
bracketing and direct anatomical evidence. Under the former approach, the discovery of
a fossil representative belonging to an entirely terrestrial clade can be deemed to provide
evidence of life on land, even when the state of preservation of the individual fossils
is not particularly impressive. The second, more direct approach, relies on identifying
unambiguous terrestrial adaptations in fossil specimens to conclude that these indeed lived
on land.

Biology 2022,11, 1516 5 of 18
Biology 2022, 11, x 5 of 19
limbs; growth through successive moults (ecdysis); and an open circulatory system with
a dorsal heart with lateral valves [55]. This set of unique characteristics suggests that they
are a monophyletic group (descendants of a common ancestor who possessed the diag-
nostic characteristics of the lineage). Arthropods are represented by chelicerates (with
arachnids such as spiders and scorpions, and marine groups such as pycnogonids and
horseshoe crabs); myriapods (such as millipedes and centipedes); hexapods (containing
insects) and predominantly aquatic ‘crustaceans’ (for example crabs and prawns), which
are collectively known as pancrustaceans; and include important extinct groups, such as
the trilobites (Figure 2). Their abundance makes arthropods ecologically essential; for ex-
ample, myriapods are important processors of detritus in forests, and termites consume
such large amounts of cellulose that they are significant for the carbon cycle and atmos-
pheric gas composition [56]. Without arthropods, life and ecosystems on Earth would be
radically different. Their surprising diversity (which exceeds 75% of all living species de-
scribed [57]) can help to elucidate the patterns and processes of macroevolution.
Figure 2. Present diversity of arthropods (A) pycnogonid Endeis flaccida (chelicerate); (B) xiphosuran
Limulus polyphemus (chelicerate); (C) spider Philodromus aureolus (arachnid: chelicerate); (D) milli-
pede Cylindroiulus caeruleocinctus (myriapod); (E) centipede Scutigera coleoptrata (myriapod); (F)
branchiopod Daphnia sp. (pancrustacean); (G) remipede Morlockia williamsi (pancrustacean); (H)
hexapod Orchesella villosa (pancrustacean). Image sources: Wikimedia Commons; (G) Jørgen Olesen.
The earliest animals we know as land-dwelling were arthropods [58]. Evaluating the
earliest fossil evidence of arthropod life on land can rely on two approaches—phyloge-
netic bracketing and direct anatomical evidence. Under the former approach, the discov-
ery of a fossil representative belonging to an entirely terrestrial clade can be deemed to
provide evidence of life on land, even when the state of preservation of the individual
fossils is not particularly impressive. The second, more direct approach, relies on identi-
fying unambiguous terrestrial adaptations in fossil specimens to conclude that these in-
deed lived on land.
Figure 2.
Present diversity of arthropods (
A
) pycnogonid Endeis flaccida (chelicerate); (
B
) xiphosuran
Limulus polyphemus (chelicerate); (
C
) spider Philodromus aureolus (arachnid: chelicerate); (
D
) millipede
Cylindroiulus caeruleocinctus (myriapod); (
E
) centipede Scutigera coleoptrata (myriapod); (
F
) branchio-
pod Daphnia sp. (pancrustacean); (
G
) remipede Morlockia williamsi (pancrustacean); (
H
) hexapod
Orchesella villosa (pancrustacean). Image sources: Wikimedia Commons; (G) Jørgen Olesen.
The earliest fossil assemblage preserving arthropods belonging to terrestrial clades is
the Pˇrídolí-aged Ludlow bone bed Member exposed at Ludford Lane, near Ludlow in Shrop-
shire, western England [
34
,
59
–
61
]. This site contains a range of myriapods
(Figure 1E),
in-
cluding scutigeromorph centipedes in the genus Crussolume [
61
], the arthropleurid Eoarthro-
pleura [
61
], and a singular specimen of the trigonotarbid arachnid Eotarbus jerami Dunlop
1996 (= Palaeotarbus jerami, junior synonymy resolved by Dunlop [
62
]; Figure 1D). Any of
these can be confidently considered to be the oldest terrestrial arthropod body fossils, albeit
the fidelity of their preservation does not permit the observation of anatomical adaptations
for life on land—most are represented by small shreds of cuticle or, in the case of Eotarbus,
a dark carbonised specimen. U-Pb zircon dating of the Ludlow bone bed at Ludford Lane
in Shropshire constrained the age of the deposit to ~420 Ma [63].
The earliest animal possessing unambiguous terrestrial adaptations is the millipede
Pneumodesmus newmani from the Lower Devonian Cowie Harbour near Stonehaven in Ab-
erdeenshire, Scotland [33], which is preserved with more fidelity. The terrestrial character
of this organism is indisputable since it possesses spiracles, openings on the cuticle that
allow air to enter the tracheal system (Figure 1F). Two other diplopod species were reported
from the locality, all described by Wilson and Anderson [
33
]. The Dictyocaris Member of
the Cowie Formation at Cowie Harbour was initially considered to be Silurian based on
palynological evidence (~426.9 Ma [
64
–
66
]), but isotopic dating confidently constrained its
age to the lowermost Devonian (Lochkovian; ~414 Ma [
67
]), making it some 6 Mya younger
than the Ludford Lane assemblage. Recently, the scorpion Palaeoscorpius devonicus [
68
,
69
]
from the Lower Devonian Hunsrück Slate Lagerstätte in Germany (~405 Ma) was inter-

Biology 2022,11, 1516 6 of 18
preted as possessing adaptations for life on land, namely probable book lungs, indicating
that it was likely terrestrial [70].
2.2. Arthropod Phylogeny
The evolutionary relationships among the major arthropod groups have always been
a subject of debate, such that by the start of the 21st century virtually all conceivable
topologies for the group had been proposed [
71
]. Identifying the closest relatives of each
terrestrial lineage is crucial, not only for comparative studies dealing with adaptation
strategies for life on land, but also to understand the potential terrestrialization routes
and constrain their timing. To infer these phylogenies, the anatomical structures of living
and fossil species provide a treasure trove of comparative data that has been expanded
even further during the last few decades by vast quantities of molecular data [
72
]. In their
adaptation to land, arthropods have undergone convergent evolution (independent origins
of similar biological systems in different lineages), which has often complicated efforts to
assess kinship relationships between them [
54
]. For example, trachea (respiratory structures
adapted to terrestrial environments) are found in several lineages that have conquered the
land independently during the Palaeozoic: in a few arachnids, myriapods, isopods, and
hexapods. The introduction of genome-scale phylogenetic analyses-phylogenomics—has
greatly narrowed down the number of hypotheses on hexapod phylogeny, but crucially,
some nodes of the arthropod tree remain difficult to resolve. Such challenging nodes
often represent ancient and rapid radiations that are complex to address with any dataset,
molecular or morphological, and represent the major lasting controversies in reconstructing
the process of the arthropod invasion of land [18,66,73–75].
2.3. Myriapods
According to a classical phylogenetic hypothesis, the exclusively terrestrial myriapods,
have been regarded as the sister group of the hexapods. This hypothetical clade, called
Tracheata (or Atelocerata), is supported mainly by the presence of tracheae in both groups
to carry out gas exchange (reviewed in [
76
]). Current studies based on molecular data, and
also a re-examination of more subtle morphological characters of the nervous system and
ommatidia [
73
,
77
], discard this hypothesis, and attribute this coincidental morphological
convergence to independent convergence [
78
]. A second hypothesis recovered by early
analyses of molecular data implicated myriapods as a sister group to the chelicerates
(Myriochelata or Paradoxopoda). However, these results are now considered as caused by
a phylogenetic reconstruction bias due to the rapid evolutionary rates of pancrustaceans
attracting to the outgroup and pushing myriapods and chelicerates into an artefactual clade
when using simpler models of molecular evolution [
79
]. Today, there is a certain consensus
on the main relationships between arthropods, supported by phylogenomic data [
78
]. The
myriapods, the first of the three large terrestrial lineages, are generally accepted as a sister
group to the pancrustaceans (hexapods and all crustacean lineages), and the chelicerates as
the closest relative of this clade (Figure 3). Thus, the basic division between arthropods
consists of those that have mandibles (myriapods and pancrustaceans) and chelicerae.
The internal phylogeny of myriapods, though, is currently more contentious. Several
recent phylotranscriptomic analyses disagree on the exact relationship between their main
lineages [
78
,
80
–
83
] but they do not have an impact on the single terrestrialization event
inferred for the group.

Biology 2022,11, 1516 7 of 18
Biology 2022, 11, x 7 of 19
morphological convergence to independent convergence [78]. A second hypothesis recov-
ered by early analyses of molecular data implicated myriapods as a sister group to the
chelicerates (Myriochelata or Paradoxopoda). However, these results are now considered
as caused by a phylogenetic reconstruction bias due to the rapid evolutionary rates of
pancrustaceans attracting to the outgroup and pushing myriapods and chelicerates into
an artefactual clade when using simpler models of molecular evolution [79]. Today, there
is a certain consensus on the main relationships between arthropods, supported by phy-
logenomic data [78]. The myriapods, the first of the three large terrestrial lineages, are
generally accepted as a sister group to the pancrustaceans (hexapods and all crustacean
lineages), and the chelicerates as the closest relative of this clade (Figure 3). Thus, the basic
division between arthropods consists of those that have mandibles (myriapods and pan-
crustaceans) and chelicerae. The internal phylogeny of myriapods, though, is currently
more contentious. Several recent phylotranscriptomic analyses disagree on the exact rela-
tionship between their main lineages [78,80–83] but they do not have an impact on the
single terrestrialization event inferred for the group.
Figure 3. Cladogram with the current consensus on the phylogenetic relationships between the main
groups of arthropods. The terrestrial groups are represented in orange colours while the marine
clades in blue and turquoise for Branchiopoda (fresh water). The thickness of the terminal branches
corresponds to a proportional approximation of the number of described species. At the base of the
cladogram, image with detail of chelicerae and mandibles, the defining structures of the two groups.
Some of the silhouettes come from Phylopic (phylopic.org/; accessed on 5 November 2022).
2.4. Pancrustacea (Hexapoda)
There is strong molecular and morphological evidence that favours the position of
hexapods as nested within the ‘crustaceans’ (as the clade Pancrustacea, or Tetraconata),
and myriapods as the sister group of pancrusteans forming the Mandibulata group, char-
acterized by the presence of this distinctive oral structure [84–86]). In contrast, the exact
relationships of hexapods within the Pancrustacea are still unclear, and it is not obvious
which is their aquatic sister group. Phylogenomic datasets have variously lent support to
the mostly freshwater-dwelling branchiopods [25], or the species-poor and enigmatic
remipedes [68,70]. Establishing which ‘crustacean’ group is the most closely related to
hexapods has a great impact on whether the latter group presumably colonised terrestrial
environments directly from the sea, or whether they first colonized freshwater environ-
ments and later moved to land. Most recent phylogenomic studies, though, using hun-
dreds of molecular markers, have shifted the balance in favour of Remipedia [84,86].
Figure 3.
Cladogram with the current consensus on the phylogenetic relationships between the main
groups of arthropods. The terrestrial groups are represented in orange colours while the marine
clades in blue and turquoise for Branchiopoda (fresh water). The thickness of the terminal branches
corresponds to a proportional approximation of the number of described species. At the base of the
cladogram, image with detail of chelicerae and mandibles, the defining structures of the two groups.
Some of the silhouettes come from Phylopic (phylopic.org/; accessed on 5 November 2022).
2.4. Pancrustacea (Hexapoda)
There is strong molecular and morphological evidence that favours the position of
hexapods as nested within the ‘crustaceans’ (as the clade Pancrustacea, or Tetraconata),
and myriapods as the sister group of pancrusteans forming the Mandibulata group, char-
acterized by the presence of this distinctive oral structure [
84
–
86
]. In contrast, the exact
relationships of hexapods within the Pancrustacea are still unclear, and it is not obvious
which is their aquatic sister group. Phylogenomic datasets have variously lent support
to the mostly freshwater-dwelling branchiopods [
25
], or the species-poor and enigmatic
remipedes [
68
,
70
]. Establishing which ‘crustacean’ group is the most closely related to
hexapods has a great impact on whether the latter group presumably colonised terrestrial
environments directly from the sea, or whether they first colonized freshwater environ-
ments and later moved to land. Most recent phylogenomic studies, though, using hundreds
of molecular markers, have shifted the balance in favour of Remipedia [
84
,
86
]. Remipedia
are a class of blind and predatory crustaceans that live in coastal aquifers that contain saline
groundwater. They were discovered less than 40 years ago [
87
], and have a very restricted
distribution, with fewer than 30 known species described from the anchialine caves in the
Caribbean Sea, two species from the Canary Islands and one from Western Australia. Very
little is known about the biology of these organisms, which makes it difficult to understand
their significance for hexapod terrestrialization.
2.5. Pancrustacea (Isopods)
The suborder Oniscidea (woodlice) represents the most diverse isopod crustacean
group, with over 3700 described species [
88
]. It is the only pancrustacean group besides the
hexapods composed almost entirely of terrestrial species; its members are found in almost
all terrestrial habitats, ranging from nearshore settings to forests [
89
]. In particular, the in-
tertidal genus Ligia inhabiting shorelines is often regarded as a transitory group [
90
]. Given
their varying degrees of adaptations for life in semi-aquatic and terrestrial environments,

Biology 2022,11, 1516 8 of 18
woodlice provide a rewarding model group for understanding the transition from marine
to terrestrial habitats, which hinges on an understanding of their phylogeny [
90
,
91
]. Mor-
phological studies implicate Ligiidae as the basalmost woodlouse clade, implying a single
invasion of land directly from the marine realm [
92
], although some molecular studies have
challenged the monophyly of the group (e.g., [
93
,
94
]). Overall, isopods remain probably the
least-studied terrestrialization event among arthropods. Their fossil record is fragmentary
and scarce, with their oldest occurrence from the Cretaceous (summarised in [
90
]). If
terrestrial isopods originated in the late Palaeozoic, potentially the Carboniferous [
90
], they
would represent the most recent arthropod terrestrialization event.
In some sense, other pancrustacean clades such as amphipods and the decapods, also
invaded semi-terrestrial habitats (e.g., supralittoral zone of beaches, most soil and leaf litter,
edges of freshwater habitats) and these have been considered as terrestrialization events
by some (e.g., [
13
]). Here, we refrain from treating these groups as fully terrestrial, since
their adaptation to life is not as developed as in the case of the woodlice. Nonetheless,
these taxa represent important study groups for future research in arthropod adaptation to
semi-terrestrial habitats.
2.6. Arachnids
Among terrestrial arthropods, only insects outnumber arachnids in terms of the num-
ber of described species (1 million versus 112,000, respectively; [
51
]). The clade Arachnida
includes all terrestrial chelicerates, composed mainly of predatory groups such as spiders
and scorpions, and parasites such as ticks. However, chelicerates also include marine
taxa such as the pycnogonids (sea spiders) and xiphosurans (horseshoe crabs). Neither
the currently available morphological nor molecular data have unequivocally resolved
the internal kinship relationships between chelicerates [
66
]. Arachnids have traditionally
been regarded as a monophyletic group, implying that a single and irreversible ancestral
colonization of land paved the way to this group’s evolutionary success. Some recent
studies including genome-scale and morphological phylogenies, however, do not support
this relationship, instead placing the marine Xiphosura within terrestrial arachnids, and
not as a sister group to it [
74
,
95
]. The focus of this debate is whether there has been a single
common ancestor for all terrestrial arachnids, a single terrestrialization event within a com-
mon ancestor of terrestrial arachnids + xiphosurans (with the later transitioning again into
aquatic environments soon after), or whether arachnid terrestrialization occurred on two or
more separate occasions. Resolving this puzzle is enormously significant, as it rewrites our
perception of the evolution of terrestrial adaptations (e.g., the respiratory system, sensory
and reproductive systems, and the locomotor appendages). The physiological demands
of life on land require a significant modification of these anatomical features, which is
probably best illustrated by the respiratory organs, a great variety of which are present in
extant chelicerates (book lungs and tracheae in terrestrial groups, and book gills in marine
forms) [
96
,
97
]. If xiphosurans were a group of marine arachnids, this may suggest that
the remaining lineages colonized land independently. A second option would be that
xiphosurans recolonized the marine environment from a terrestrial ancestor. Of these two
options, the first would be considered more plausible, since the fossil record of Xiphosura
extends back more than 400 Ma with exclusively aquatic forms, without traces of a potential
terrestrial or amphibious ancestors [
98
] (Figure 1D). Furthermore, no widespread losses
of terrestrial respiratory organs in arthropods are known, once acquired, in line with the
predictions of Dollo’s law [99].
In addition, even though horseshoe crabs can make momentary incursions into the
coasts to spawn eggs, they do not have distinctly terrestrial morphological adaptations
and their body structures present great similarity, and probably homology, with that
of other aquatic fossil chelicerates [
100
,
101
]. Other recent studies using genome-scale
datasets, as well as morphological and fossil evidence suggest that marine chelicerates
(pycnogonids and Xiphosura) are successive sister groups of a monophyletic lineage of

Biology 2022,11, 1516 9 of 18
terrestrial arachnids. These results are compatible with a single colonization of land within
chelicerates and the absence of wholly marine arachnid orders [66,102].
3. Pre-Devonian Fossil Record of Terrestrial Arthropods
3.1. Trace Fossil Evidence
The oldest traces of activity in the terrestrial environment made by arthropods (ichno-
fossils) date from the middle Cambrian to the Early Ordovician. The oldest of these include
trackways on land from the late Cambrian (~500 Ma) of Ontario, Canada produced by
arthropods with at least 11 pairs of similar walking legs and a long tail-spine, presumably
made by the extinct euthycarcinoids [
103
] (Figure 1E). Another site famous for its Cambrian
traces of life on land is the middle to late Cambrian Blackberry Hill in central Wisconsin,
which preserves diversity of arthropod trackways in a tidal flat and nearshore environment,
along with the remains of the oldest euthycarcinoid, Mosineia [
104
]. Massive trackways
from the intertidal zone left by euthycarcinoids with walking legs during the Cambrian and
Ordovician indicate longer stays on land where these amphibious animals may have come
in pursuit of shallow lagoons and freshwater pools [
105
], albeit their excursions on land
may have been short-lived [
106
]. Other early arthropods to make temporary excursions to
near-shore habitats were the trilobites, whose trace fossils in tidal-flat deposits are known
since the Cambrian, albeit their traces were likely made subaqueally [
107
,
108
]. Trilobites
possessed gill lamellae for respiration [
109
,
110
], which are unlikely to have provided them
with the ability to survive on land for prolonged periods of time. Myriapods have been
implicated in producing Ordovician backfilled burrows from Pennsylvania (445 Ma [
111
]),
although the terrestrial nature of this deposit has been later disputed [
36
]. A slightly
younger record of trackways and trails (Diplichnites and Diplopodichnus) from Cumbria,
England (>450 Ma) records myriapods moving alongside the edges of ponds, but these
were likely made under water [
112
,
113
]. Overall, locomotive traces documented through-
out the Cambrian and Ordovician reinforce the view that aerial activities of arthropods (if
not terrestrial arthropods) were common on the coasts and along the edges of freshwater
bodies during this time.
3.2. Body Fossil Evidence
The availability of land-dwelling arthropod body fossils is fundamentally constrained
by the limited number of terrestrial formations before the Devonian and the limited interest
these geological units have attracted in the past [
42
]. Fossils generally require a steady
rate of sedimentation to preserve, which is not an easily achievable condition for minute
soft-bodied arthropods inhabiting the soil or decaying vegetation matter. As such, palaeon-
tologists have to rely on a restricted set of fossil localities that provide unusual preservation
windows for their time.
The earliest relatives of myriapods in the fossil record are the Cambrian to Triassic
euthycarcinoids, mentioned earlier for the terrestrial trace fossils. The affinities of this group
have been traditionally difficult to pinpoint, but recent findings of exceptionally preserved
Devonian specimens establish the group as the stem-group to myriapods [
39
]. These
aquatic arthropods were amphibious, ranging from marine and brackish to freshwater
deposits [
114
]. Their ventures on land have been variously interpreted as short migrations
between ephemeral freshwater pools, grazing on microbial mats and detritus, or migrations
to fertilise eggs on land like in modern horseshoe crabs [
103
,
115
]. Recent synchrotron
studies revealed probable respiratory organs in a Devonian euthycarcinoid, consistent with
an amphibious lifestyle [
116
]. Other early myriapod remains are known from the Silurian
Kerrera (425 Ma) and Ludlow (420 Ma) deposits in the United Kingdom, albeit it is difficult
to determine if they were truly terrestrial [
59
,
117
]. The earliest undoubtedly terrestrial
fossil myriapod is the millipede Pneumodesmus newmani from Cowie in Scotland, originally
regarded as Late Silurian [
33
], but more recently as Lower Devonian (414 Ma [
67
]). Its
terrestrial ecology is indicated by the presence of spiracles.

Biology 2022,11, 1516 10 of 18
The earliest hexapod fossils are the Early Devonian (~405 Ma) springtail Rhyniella
praecursor [
118
,
119
] and the enigmatic Leverhulmia mariae [
120
], from the coeval Rhynie
and Windyfield chert deposits in Scotland, which became preserved with extraordinary
fidelity when silica-rich water from volcanic springs inundated hot springs and the sur-
rounding land. While various systematic positions of the peculiar Leverhulmia have been
proposed, Rhyniella is a crown-group springtail, not that different from species that in-
habit soil and leaf litter today [
121
], suggesting that this clade of hexapods radiated well
before the Early Devonian. Nonetheless, insect fossils before the Carboniferous are few;
the Rhynie chert is followed by a window of 80 Ma (referred to as the ‘hexapod gap’)
during which no insects are known [
122
]. The existence of pre-Devonian hexapods is a
reasonable assumption, proposed already by early cladistic studies predating the molec-
ular clock methodology [
123
]. Although a decade-old bounty of 1000 dollars has been
put on an undisputable insect fossil from the pre-Devonian [
124
], this sum remains to
be claimed. Instead, the hunt for early hexapods yielded a number of dubious records,
like fossils only seen once and never again [
125
], suspected modern contaminants [
106
],
and miss-identifications, such as purported Devonian insect wings that turned out to be
malacostracan tail fans [
126
,
127
]. A recent review is provided by [
128
]. Others represent
genuinely difficult fossils to interpret, such as the purported Devonian hexapod Strudiella
devonica [
129
], which may however represent a decayed non-insect arthropod [
130
], or
the Devonian Wingertshellicus/Devonohexapodus at once interpreted as an aquatic stem-
hexapod [
131
], but not unequivocally accepted [
132
]. It is interesting to note that even
in deposits such as the Rhynie chert where arthropod cuticles are not rare in some facies,
the vast majority belong to arachnids, not hexapods as may be expected from modern
ecosystems, where insects predominate. Winged insects only came to dominate terrestrial
ecosystems by the Carboniferous, leading many to postulate that hexapods may have been
species poor until the origin of with wings [
42
] that appear unequivocally in the fossil
record in the latest Mississippian (~322 Ma [133]).
Among arachnids, we find the oldest fossil evidence of arthropod life on land, repre-
sented by scorpion remains from the Silurian (~437 Ma [
134
]). However, their terrestriality
is not unambiguous due to the absence of bona fide terrestrial characters, such as book
lungs, and have been found in aquatic or semi-aquatic deposits [
135
,
136
] (Figure 1H). Puta-
tive book lungs have been reported from a fossil scorpion from the Devonian Hunsrück
Slate in Germany (~405 Ma [
70
]). Current molecular, phylogenomic and morphological
evidence suggests, however, that scorpions are arachnids related to spiders [
102
,
137
,
138
],
in a clade of mostly lung-bearing arachnids known as Arachnopulmonata. Within this
clade, the latest phylogenomic results suggest that pseudoscorpions are the closest relatives
of scorpions [
66
,
74
,
139
]. This phylogenetic position is hardly reconcilable with a marine
origin of scorpions, suggesting that some of these ancestral scorpions may have secondar-
ily returned to the aquatic environment, although without obvious marine adaptations.
The earliest member of Trigonotarbida, a group of extinct terrestrial arachnids known to
possess book lungs [
140
,
141
], is known from the Silurian (~420 Ma [
63
]) Ludford Lane
in England [
59
]. Trigonotarbids persisted until the Permian and are known in stunning
anatomic detail, in part thanks to their preservation in Rhynie chert [142]).
4. Reconciling Rocks and Clocks
4.1. Methodologies to Build Chronologies
The abundant arthropod fossil record is informative on the diversity of the group,
the historical evolution of morphological characters, and provides temporal guidelines for
molecular dating. Solving the relative times of evolutionary divergences between species
and clades in the geological past provides crucial information for dating the origin of terres-
trial ecosystems. The reconstruction of these “timetrees”, or chronograms, is increasingly
methodologically sophisticated and has become the backbone for comparative studies of
evolutionary biology and palaeontology. Molecular data inform us both on the understand-
ing of the tree’s branching pattern (the phylogeny) and, once calibrated with fossils, on

Biology 2022,11, 1516 11 of 18
the timing at which these branching events occurred (the timeline). The dates are inferred
using the molecular clock technique [
143
], where the time elapsed since the divergence of
different organisms or species is deduced from the differences between their DNA or amino
acid sequences. To carry out these analyses, calibration points are routinely used where
minimum ages are defined based on the oldest fossil evidence that can be unequivocally
assigned to that node, that is, the origin of that group cannot be younger than its oldest
fossil [
144
]. Node dating is the most widely used method [
145
], and it has developed a
lot in recent years, with the implementation of Bayesian methods that allow assigning
probabilities to age ranges and to other various parameters based on previous knowledge
about the group in question [
146
]. While the chronologies constrain the real age of the lin-
eages, the fossils inform us of when those organisms became numerically and ecologically
abundant. Furthermore, including fossils in phylogenetic analyses helps arrive at more
accurate trees and divergence time estimates [
147
–
149
]. Therefore, chronologies provide
an essential conceptual framework for investigating the evolution of the first terrestrial
ecosystems and the interactions over time between organisms and their environment.
4.2. Dating the Arthropod Terrestrialization
Most recent chronologies of arthropod radiation (or subgroups of them) using molecu-
lar clocks are generally compatible with paleontological evidence, proposing an origin of
the group between the end of the Ediacaran period and the beginning of the Cambrian (with
credibility intervals falling with 95% of probability between 551–536 Ma) [
37
]. These studies
also suggest the origin of arachnids and hexapods are in some consensus with the fossil
evidence, preceding the oldest fossils by a few tens of thousands of years (Figure 4). In the
case of chelicerates, the origin of terrestrial arachnids and of their main diversifications have
been inferred to fall between the Cambrian and Ordovician (494–475 Ma) [
66
]. Molecular
evolution rates were likely high during its origin, coinciding with a rapid cladogenesis [
37
].
When xiphosurans are nested within arachnids, the origin of this clade is inferred in ages
comprising mostly Ediacaran [
95
] to Cambrian period [
66
]. For hexapods, the estimated
ages vary in different studies between 520–450 Ma (summarised in [
150
]). Likewise, a
Cambrian–Ordovician origin has been proposed for myriapods [25,78,83,151–153].
Consequently, there are certain differences when ages inferred from molecular dating
studies are compared with the oldest fossil record, where arachnids first appeared in the
Silurian (427 Ma) and hexapods in the Devonian (411 Ma). In the case of arachnids, it has
been suggested that these differences may be due to the fact that the closest relative of
arachnids is an extinct group. Eurypterids (also called ‘sea scorpions’) have been proposed
as a possible sister group (Figure 1G). These aquatic organisms emerged during the Or-
dovician (~467 Ma) and represented an important component of marine fauna until they
disappeared from the fossil record during the end-Permian mass extinction (~252 Ma) [
10
].
It seems that they could make inroads into the terrestrial environment, as suggested by
ichnofossils, and recent studies show that they had respiratory structures adapted to breath-
ing air, possibly since the Cambrian–Ordovician [
40
]. The latter study suggests that their
ancestor may have been semi-terrestrial, similar to eurypterids. Regarding the origin of
myriapods, the divergence times inferred are substantially older (524–505 Ma) than their
oldest fossil evidence, and they firmly place the earliest members of this group in the
Cambrian [
37
,
78
,
151
–
153
], despite the fact that its oldest fossil is 414 Ma (Figure 4). The
reinterpretation of Euthycarcinoidea as the closest relative of myriapods based on the simi-
larity of mouth and eye structures bridges this gap between the fossil record and molecular
clocks [39].

Biology 2022,11, 1516 12 of 18
Biology 2022, 11, x 12 of 19
Figure 4. Schematic chronogram with divergence times between the most representative arthropod
clades. The internal nodes of the tree fall into the mean estimated divergences taken from recent
studies cited in the text. On the x-axis, time runs from most recent (right) to the past (left), and is
expressed in millions of years. The yellow rectangles mark the credibility intervals for the different
terrestrialization phenomena. The dagger symbol represents the oldest known fossil in that terres-
trial group, the double dagger represents the oldest direct evidence of terrestrial breathing struc-
tures, and the asterisk the oldest trace evidence of terrestrial behaviour. The terrestrial groups are
represented in orange colours while the marine clades in blue and turquoise for Branchiopoda (fresh
water). Some of the silhouettes are from Phylopic (phylopic.org/; accessed on 5 November 2022).
Consequently, there are certain differences when ages inferred from molecular da-
ting studies are compared with the oldest fossil record, where arachnids first appeared in
the Silurian (427 Ma) and hexapods in the Devonian (411 Ma). In the case of arachnids, it
has been suggested that these differences may be due to the fact that the closest relative
of arachnids is an extinct group. Eurypterids (also called ‘sea scorpions’) have been pro-
posed as a possible sister group (Figure 1G). These aquatic organisms emerged during the
Ordovician (~467 Ma) and represented an important component of marine fauna until
they disappeared from the fossil record during the end-Permian mass extinction (~252
Ma) [10]. It seems that they could make inroads into the terrestrial environment, as sug-
gested by ichnofossils, and recent studies show that they had respiratory structures
adapted to breathing air, possibly since the Cambrian–Ordovician [40]). The latter study
suggests that their ancestor may have been semi-terrestrial, similar to eurypterids. Re-
garding the origin of myriapods, the divergence times inferred are substantially older
(524–505 Ma) than their oldest fossil evidence, and they firmly place the earliest members
of this group in the Cambrian [37,78,151–153], despite the fact that its oldest fossil is 414
Ma (Figure 4). The reinterpretation of Euthycarcinoidea as the closest relative of myria-
pods based on the similarity of mouth and eye structures bridges this gap between the
fossil record and molecular clocks [39].
Figure 4.
Schematic chronogram with divergence times between the most representative arthropod
clades. The internal nodes of the tree fall into the mean estimated divergences taken from recent
studies cited in the text. On the x-axis, time runs from most recent (right) to the past (left), and is
expressed in millions of years. The yellow rectangles mark the credibility intervals for the different
terrestrialization phenomena. The dagger symbol represents the oldest known fossil in that terrestrial
group, the double dagger represents the oldest direct evidence of terrestrial breathing structures, and
the asterisk the oldest trace evidence of terrestrial behaviour. The terrestrial groups are represented in
orange colours while the marine clades in blue and turquoise for Branchiopoda (fresh water). Some
of the silhouettes are from Phylopic (phylopic.org/; accessed on 5 November 2022).
4.3. Reconciling the Fossil and Molecular Evidence
The discrepancies between the results derived from molecular clocks and the oldest
fossil evidence may be related to the nature of the rock record, especially to the rarity
of terrestrial sediments from the Cambrian to the Silurian. It has been suggested that
Euramerica, the region from which much of the data on the first terrestrial arthropods
and plant megafossils are derived, is almost absent from terrestrial sediments before the
upper Silurian and that these are not more widespread until the Early Devonian [
50
]. This
temporal bias in the rock record possibly affects the fossil record of terrestrial organisms
and may explain part of the mismatch between molecular and fossil dates. The discrepancy
may also be explained by failures in the molecular clock methodology, particularly with
the node dating strategy. A recent method has been developed to estimate divergence
time in a total-evidence framework, where fossils are directly integrated into the combined
analysis of molecular data from living species with morphological data from fossils and
living groups [
154
]. In the process of reconstructing kinship relationships and dating
them, fossils are incorporated without having to determine their phylogenetic position
a priori, and therefore this phylogenetic uncertainty can be directly integrated in the
analysis. Some studies suggest that this approach improves divergence time estimates [
147
].
Computational limitations currently limit the application of this methodology to determine

Biology 2022,11, 1516 13 of 18
deep divergences. However, the field is advancing rapidly, and it is predicted that soon
these methodologies will help to establish the affinity of fossils, and more carefully assign
the age of the lineages and the different terrestrialization processes [155].
5. Conclusions
Ephemeral terrestrial habitats have existed for at least 1 billion years. However, animal
terrestrialization and the consequent formation of more complex habitats has been a much
more recent process. How recent remains a point of contention. The fossil record provides
the only direct source of data to understand the temporal acquisition of characters, while
phylogenies and molecular clocks complement this record to constrain the timing of the
origin of these groups. The most recent molecular dating suggests that land plants were
already present in the middle Cambrian to Early Ordovician [
38
], although other recent
molecular clock estimates push this date back even further, into the Precambrian [
156
].
Similarly, recent molecular dating studies also suggest a concomitant colonization of the
land by arthropods. If myriapods and arachnids really colonized the terrestrial environment
so early, it would be possible that millipedes, a group of detritivore myriapods, fed on
bacterial mats on the shoreline. Arachnids are a predominantly predatory group, suggesting
that they must have originated from a diverse ecosystem. In this scenario, arachnids could
have myriapods as potential prey. These ecologies represent habitats highly unfavourable
to fossilization, such as high-energy environments characterized by erosion rather than
deposition [
157
]. It is not surprising, then, that direct palaeontological insights may be
limited in these cases, and molecular inference can step in to fill the gap.
Author Contributions:
Conceptualization, J.L.-F.; writing—original draft preparation, J.L.-F., E.T.,
R.J.H. and C.C.; writing—review and editing, J.L.-F., E.T., R.J.H. and C.C. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was funded by the Strategic Priority Research Program of the Chinese
Academy of Sciences (XDB26000000), the National Natural Science Foundation of China (42288201,
42222201), and the Second Tibetan Plateau Scientific Expedition and Research project (2019QZKK0706).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
We are grateful to Greg Edgecombe, who provided constructive comments on a
previous version of this manuscript, as well as three anonymous reviewers. We would like to thank
the authors of images deposited in open access that have been used to generate some of the figures.
Conflicts of Interest: The authors declare no conflict of interest.
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