Non-ammocoete larvae of Palaeozoic stem lampreys

Abstract

Ammocoetes—the filter-feeding larvae of modern lampreys—have long influenced hypotheses of vertebrate ancestry1–7. The life history of modern lampreys, which develop from a superficially amphioxus-like ammocoete to a specialized predatory adult, appears to recapitulate widely accepted scenarios of vertebrate origin. However, no direct evidence has validated the evolutionary antiquity of ammocoetes, and their status as models of primitive vertebrate anatomy is uncertain. Here we report larval and juvenile forms of four stem lampreys from the Palaeozoic era (Hardistiella, Mayomyzon, Pipiscius, and Priscomyzon), including a hatchling-to-adult growth series of the genus Priscomyzon from Late Devonian Gondwana. Larvae of all four genera lack the defining traits of ammocoetes. They instead display features that are otherwise unique to adult modern lampreys, including prominent eyes, a cusped feeding apparatus, and posteriorly united branchial baskets. Notably, phylogenetic analyses find that these non-ammocoete larvae occur in at least three independent lineages of stem lamprey. This distribution strongly implies that ammocoetes are specializations of modern-lamprey life history rather than relics of vertebrate ancestry. These phylogenetic insights also suggest that the last common ancestor of hagfishes and lampreys was a macrophagous predator that did not have a filter-feeding larval phase. Thus, the armoured ‘ostracoderms’ that populate the cyclostome and gnathostome stems might serve as better proxies than living cyclostomes for the last common ancestor of all living vertebrates.

Full text

408 | Nature | Vol 591 | 18 March 2021
Article
Non-ammocoete larvae of Palaeozoic
stem lampreys
Tetsuto Miyashita1,2,3,4,7 ✉, Robert W. Gess5,7, Kristen Tietjen1,6 & Michael I. Coates1
Ammocoetes—the lter-feeding larvae of modern lampreys—have long inuenced
hypotheses of vertebrate ancestry1–7. The life history of modern lampreys,
whichdevelop from a supercially amphioxus-like ammocoete to a specialized
predatory adult, appears to recapitulate widely accepted scenarios of vertebrate
origin. However, no direct evidence has validated the evolutionary antiquity of
ammocoetes, and their status as models of primitivevertebrate anatomy is uncertain.
Here we report larval and juvenile forms of four stem lampreys from the Palaeozoic era
(Hardistiella, Mayomyzon, Pipiscius, and Priscomyzon), including a hatchling-to-adult
growth series of the genus Priscomyzon from Late Devonian Gondwana. Larvae of all
four genera lack the dening traits of ammocoetes. They instead display features that
are otherwise unique to adult modern lampreys, including prominent eyes, a cusped
feeding apparatus, and posteriorly united branchial baskets. Notably, phylogenetic
analyses nd that these non-ammocoete larvae occur in at least three independent
lineages of stem lamprey. This distribution strongly implies that ammocoetes are
specializations of modern-lamprey life history rather than relics of vertebrate ancestry.
These phylogenetic insights also suggest that the last common ancestor of hagshes
and lampreys was a macrophagous predator that did not have a lter-feeding larval
phase. Thus, the armoured ‘ostracoderms’ that populate the cyclostome and
gnathostome stems might serve as better proxies than living cyclostomes for the last
common ancestor of all living vertebrates.
In evolutionary narratives of vertebrate origin, ammocoetes remain
a convenient model—if not an analogue—of hypothetical ancestral
states, because few alternatives are offered by the controversial fossil
forms attributed to the vertebrate stem or the anatomically specialized
living chordates8. The overall resemblance between ammocoetes and
cephalochordates drove early comparative analyses to postulate an
ammocoete-like, sand-burrowing filter feeder at the base of vertebrate
phylogeny
2–5
, and even to propagate the idea that cephalochordates
are ‘degenerate’ vertebrates
1
. Circumstantial support for these sce-
narios has begun to erode in recent times. Cephalochordates have now
been removed to the earliest branch of the chordate crown9; hagfish,
which have now been shown to be firmly allied as the sister group to
lampreys
10
, show no trace of an ammocoete-like larval phase
11
; and puta-
tive stem vertebrates (Metaspriggina and myllokunmingiids) from the
Cambrian period are morphologically remote from ammocoete-like
conditions (such as fused branchial arches or primordial sensory cap-
sules)
12,13
. However, in the absence of direct contradictory evidence,
ammocoete-like hypothetical ancestors persist in modern hypotheses
of vertebrate origin
14–18
. These models both predict and require a deep
evolutionary origin of ammocoete morphology.
Here we present an analysis of ontogenetically immature specimens
of four Palaeozoic stem lampreys to test whether an ammocoete-like
phase existed early in the lamprey lineage. To date, the taxon Mesomyzon
mengae from Early Cretaceous lake beds of China represents the earli-
est known record of the ontogenetic transition from ammocoete to
adult19. However, the stem taxa reported here are at least 180million
years older than Mesomyzon and all of them derive from non-freshwater
environments. These exceptionally well-preserved
20
specimens show
no distinctive ammocoete-like traits. Instead, these specimens exhibit
characteristics that are associated with the adult phase of modern lam-
preys. In each fossil taxon, the immature individuals signal a transfor-
mation series that does not parallel the ontogeny of modern lampreys.
Priscomyzon riniensis, which is from the latest Devonian stage (the
Fammenian) of Waterloo Farm (South Africa), provides the earliest
and most complete ontogenetic series among the four stem lampreys
that we examined. In the original description21, the holotype (acces-
sion code: Albany Museum (AM) 5750) is the only specimen reported,
and it remains the sole representative of the adult stage in our newly
reconstructed series (Fig.1a, b, Extended Data Fig.2a–g). AM5750 has
prominent eyes22 (which were originally interpreted as otic capsules21),
a well-developed oral funnel with an annular cartilage, a ring of 14cusps
in the circumoral feeding apparatus, and 7pairs of branchial arches.
Seven additional specimens of the same taxon are reported here for
the first time. These come from the same locality as the holotype and
represent younger ontogenetic stages (Fig.1d–q). The smallest of these
(AM5813, which has a body length of 14mm) carries an abdominal bulge
https://doi.org/10.1038/s41586-021-03305-9
Received: 14 January 2020
Accepted: 28 January 2021
Published online: 10 March 2021
Check for updates
1Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL, USA. 2Canadian Museum of Nature, Ottawa, Ontario, Canada. 3Department of Biology, University of
Ottawa, Ottawa, Ontario, Canada. 4Department of Earth Sciences, Carleton University, Ottawa, Ontario, Canada. 5Albany Museum and the Department of Geology, Rhodes University,
Makhanda, South Africa. 6Biodiversity Institute & Natural History Museum, University of Kansas, Lawrence, KS, USA. 7These authors contributed equally: Tetsuto Miyashita, Robert W. Gess.
✉e-mail: TMiyashita@nature.ca

Nature | Vol 591 | 18 March 2021 | 409
of low tissue density (Fig.1p, q) that extends from behind the branchial
basket to the anal region. We tentatively identify this structure as a yolk
sac, and therefore interpret AM5813 as a hatchling.
From hatchling to adult, all eight specimens of Priscomyzon (Fig.1a–q,
Extended Data Figs.1–5) exhibit prominent eyes and anteroposteriorly
short branchial baskets that are fused at the pericardial region. Tectal
cartilages are partially preserved in the four youngest specimens, and
a funnel with the circumoral feeding apparatus is present in all but one
specimen (AM5817), which is missing the snout. In the immature indi-
viduals (AM7538 and AM5813) the feeding apparatus consists of 14pet-
aliform units, whereas in the adult (AM5750) the apparatus is preserved
as a ring of 14cusps that resembles the circumoral keratinous teeth of
modern adult lampreys. All of these characters (a circumoral feeding
apparatus, prominent eyes, tectal cartilages, and posteriorly closed
branchial baskets) occur in the adult phase of modern lampreys, but
never prior to metamorphosis23. In ammocoetes, the sensory capsules
are underdeveloped (for example, ophthalmic development is arrested
in the form of eyespots); the upper lips form a hood rather than a funnel;
the branchial baskets are long with independent left and right sides
that are aligned in parallel; and tectal cartilages are absent (Fig.1r, s).
A unique sequence of morphological changes across ontogeny
is evident in the Priscomyzon series. The diameter of the oral funnel
appears to increase in positive allometry against body width, whereas
the elongate snout becomes progressively shorter. The branchial bas-
kets also increase in proportion as the branchial arches become radially
arranged. In the adult (AM5750), the first two branchial arches extend
anterior to the eye, whereas they are immediately posterior toor level
withthe eye in younger individuals (Fig.1h–k). Although the juvenile
specimen (AM7538) is similar to the adult in discrete morphological
characters, it is no larger than the larvae—and may even be shorter in
body length than one larva (AM5817). However, a composite size trait
(the first principal component of 13 metric traits) corroborates the
ac cfa
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Fig. 1 | A growt h series of Priscomyzon riniensis. Th is growth seri es has no
ammocoe te phase, and is sh own in reverse onto genetic order. a, b, Adult stage ,
represen ted by the holoty pe AM5750, shown as a pho tograph (a) and
interpret ive drawing (b). The an atomy is reinter preted from the or iginal
description21 (‘A-4. Additional d escription s’ section o ftheSupplementar y
Infor mati on). c, Schematic re construc tion of a part of th e circumoral feedi ng
apparatus of Priscomyzon, showing t wo petaliform pla tes and cusps f rom
oblique ventr al view from a cen tre of the oral funne l. d, e, Juvenile stage,
represen ted by AM7538. This s tage is charac terized by a well- developed oral
funnel and b ranchial regio n. f–k, Late larval st age, represen ted by AM5816
(f, g), AM5815 (h, i), and AM7539 (j, k). This s tage is interm ediate in snout
length an d branchial expan sion. l–o, Early lar val stage, repre sented by AM5817
(l, m) and AM5814 (n, o). The larvae are sle nder and elonga te, and the branch ial
region is sm all compared to the r est of the body. p, q, Hatchlin g stage,
represen ted by AM5813. T his 14-mm-long spe cimen is charac terized by an
abdominal b ulge (which we identif y as a potentia l yolk sac) andhas an elongat e
snout and sma ll branchial reg ion. Prominen t eyes and a circumora l feeding
apparatusare alre ady present in t his individual a s in later ontogen etic stages .
r, s, Early ontogenetic p hases of the mo dern lamprey Petromyzon marinus.
A hatchling ( Tahara’s stage 26) (r) and an early ammoco ete (Tahara’s stage 30)
(s) in left lateral v iew. Unlike in the stem lam prey, larvae of modern l ampreys
have an oral hood , primordial eye spot s, and elongate b ranchial basket s. Scale
bars, 2mm. ac, an nular cartila ge; bb, branchial basket ; bl, bi-lobed per icardial
struct ure; cbb, pericardi al commissure b etween branc hial baskets; cfa ,
circumoral fe eding apparatu s; dt, digestive tra ct; e, eye; el, eye lens; es, eye
spot; ht, hea rt; nc, notoch ord; nhp, nasohypoph seal organ; oc, o tic capsule; of,
oral funnel; oh , oral hood; pbl, pos tbranchial lat eral structu re; pc, parachordal
commissu re; pos, perioc ular structur e; ps, piston syste m; tc, tectal c artilage;
tr, trabecular c artilage; ys, yolk sa c. Arabic nume rals denote bran chial arches,
and Roman numerals indicate elements of the circumoral feeding apparatus.

410 | Nature | Vol 591 | 18 March 2021
Article
ontogenetic sequence that was reconstructed from discrete morpho-
logical characters, and places AM7538 between the adult and larval
specimens (Extended Data Fig.9b, c). This is consistent with modern
lamprey ontogeny, in which body size decreases across the larva–juve-
nile transition (the largest ammocoetes reach nearly 1.5times the body
length of the smallest adults)24.
Other Palaeozoic stem lampreys also provide evidence of
non-ammocoete larvae. We identified two specimens of Pipiscius
zangerli that carry a yolk sac (Fig.2a–d) from the Mazon Creek fauna
(dating to the Moscovian age from the Pennsylvanian subperiod of
Illinois). The smaller of the pair (accession code Royal Ontario Museum
(ROM) 56679) has a body length of 19mm and carries a proportionally
larger sac than that of FMNH (Field Museum of Natural History) PF16082
(which has a body length of 32mm). As with Priscomyzon, these hatch-
lings are unlike ammocoetes. Both ROM56679 and FMNHPF16082 have
prominent eyes, an oral funnel, and small branchial baskets relative to
body length. Despite their diminutive size and association with a yolk
sac, the character suites of these specimens are otherwise similar to
those of larger specimens of Pipiscius. Most diagnostically for this
taxon, these characters include a feeding apparatus that is shown here
to consist of concentric rings of petaliform plates and cusps (Fig.2e,
f, Extended Data Fig.6a–h), similar to (but more numerous and prob-
ably more complex than) that of Priscomyzon. These similarities sig-
nal the affinity of Pipiscius among stem lampreys, corroborating the
original taxonomic assignment
25
that has long lacked unambiguous
morphological support
10,26,27
. A corollary of this comparison is that
such plates and cusps probably co-occurred in the feeding apparatus
of Priscomyzon and are differentially exposed or preserved in individual
specimens (Fig.1c).
One larval specimen of the stem lamprey Mayomyzon pieckoensis28
(FMNH PF8167) is known from the same locality as Pipiscius. FMNH
PF8167 is approximately one third of the length of the largest known
specimen of Mayomyzon (Fig.3) and corresponds to the late larva in
the Priscomyzon series (Fig.1h, i). Consistent with the larvae of Prisco-
myzon, it has prominent eyes, tectal cartilages, and a well-developed
oral funnel. In comparison to other specimens of Mayomyzon
29
, the
branchial region is proportionally greater in the larva (Extended Data
Fig.6i–v). The Mayomyzon series also contains specimens from sev-
eral ontogenetic stages in which the gut imprint is independent from
the branchial region (Extended Data Fig.6i–v). This trait differs from
modern ammocoetes, in which the branchial region communicates
directly with the gut. In modern lampreys, the gut becomes separated
from the branchial passage only during metamorphosis, which enables
suctorial pumping in adult lampreys30. This ontogenetically terminal
state in modern lampreys is probably the general condition among
the Palaeozoic stem taxa, regardless of ontogenetic stage, because
its correlate—a dorsally positioned gut or posteriorly joined branchial
basket—is observed in all four taxa described in this Article (Extended
Data Figs.2–7).
Furthermore, we identified new specimens to supplement a previ-
ously described series of late ontogenetic stages
31–33
in Hardistiella
montanensis, a stem lamprey from the Bear Gulch fauna (dating to the
Bashkirian age from the Pennsylvanian of Montana). The smallest speci-
men of Hardistiella (CM (Carnegie Museum of Natural History)4505)
(Extended Data Fig.7a, b) is approximately two thirds the estimated
length of the largest (CM63079 and UMPC (University of Montana
Paleontological Collections)10210), and has previously been described
as a late-stage larva
31
. Including the two new specimens (ROM78122 and
UMPC10210), the Hardistiella series shows decreasing body depth with
growth, which culminates in an anguilliform profile similar to that of
modern adult lampreys (Extended Data Figs.7a–l, 9f).
Ontogenetic transformations inferred from these Palaeozoic stem
lampreys point consistently to the absence of an ammocoete phase.
Although the Palaeozoic larval specimens are larger than modern
lamprey hatchlings (which are approximately 10mm in length), the
e
a
b
c
d
f
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inlls
Cusp
inlls
e
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e
tc?
of
cfa
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ys
an
ba1234ba
bb
oc?
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Fig. 2 | Hatc hlings of Pipis cius zangerli. The se specimen s show anatomical
traits com parable to thos e of Priscomyzon. a–d, ROM56679 (a, b) and
FMNHPF16082 (c, d) have a yolk sa c and a circumoral fee ding apparatus t hat
consist s of petaliform un its. e, f, Three-dimensional micro-computed
tomograp hy renderings s how part and coun terpart of the c ircumoral feedin g
apparatus i n a mature specim en of Pipiscius (FMNHPF8344). Eac h petaliform
plate is split i nto lobes by a long itudinal groove towa rd the outer edge. At l east
three (and pote ntially four) rings of cu sps are preser ved (those demarc ating
the anteri or-most plate are highlig hted), and each cus p occurs at the pla te
boundari es. Scale bar s, 2mm. an, anus; ba, bran chial arch; other abb reviations
as in Fig.1.
a
b
1234567
bb
e
e
tc
ac? dt pcs?
oc
of tr
Fig. 3 | Lar val specime n of M.pieckoensis (FMNH PF8167). T his specimen
shows anatom ical traits co mparable to thos e of Priscomyzon: it has an elon gate
snout, promin ent eyes, and sep aration bet ween digestive t ract and branc hial
passage . Scale bar, 2mm. pcs, per icardial struc ture; other abbr eviations as in
Fig.1.

Nature | Vol 591 | 18 March 2021 | 411
former are sufficiently small as to make a preceding ammocoete phase
extremely unlikely. Otherwise, ammocoete traits would have to develop
early, only to metamorphose at diminutive size into the forms repre
-
sented by AM5813 and ROM56679 (Figs.1n, o, 2a, b). By contrast, living
lampreys persist as filter-feeding ammocoetes for 2–7years, grow
much larger than these Palaeozoic larvae, and usually metamorphose
at lengths of 150–250mm
24
. Although no hatchlings have been identi-
fied in Hardistiella and Mayomyzon, the similarities that are manifest
in the later ontogenetic stages add support to the generality of the
ontogenetic pattern thatwe observed in Priscomyzon (Extended Data
Fig.9). This reconstructed life history for the stem lampreys appears
to be consistent with palaeoecological settings. All three localities
represent protected, shallow, marine-influenced habitats in which
larvae, juveniles, and sexually mature and/or gravid adults of other
fishes (placoderms, chondrichthyans, actinopterygians, and sarcop-
terygians) occur
34–38
. It is difficult to determine whether these stem
lampreys represent direct or indirect developers
13
. In Priscomyzon,
immature and adult forms are set apart by several characters (Extended
Data Fig.9c), and the transition may therefore qualify as metamorpho-
sis. A possible reduction in body length across this transition is also
reminiscent of metamorphosis in modern lampreys
24
(Extended Data
Fig.9b). However, there is no clear evidence for the marked dietary
shift that is usually associated with metamorphosis.
With the addition of these Palaeozoic stem lampreys, the results of
our phylogenetic analysis support a reverse character polarity to the
prediction of ammocoete-based hypotheses of vertebrate origins:
that is, the life history of modern lampreys represents a derived con-
dition39,40. Life-history traits that are distinct from those of modern
lampreys—including large hatchling size relative to the adult, and the
lack of evidence for filter-feeding larvae—occur in three stem-lamprey
branches and successive outgroups of the lamprey clade(Fig.4). This
character distribution offers two further implications: (a) similar to
hagfish and modern adult lampreys, the last common ancestor of living
cyclostomes was probably a macrophagous predator; and (b) condi-
tions at the crown vertebrate node might be better informed by the
armoured taxa that occupy both the cyclostome and gnathostome
stems (namely, the ostracoderms) (Extended Data Fig.10). Thus, the
cyclostome and gnathostome crowns each represent an independ-
ent evolution of carnivory that emerged from the stem assemblage
of deposit feeders such as anaspids, galeaspids, osteostracans, and
thelodonts7.
Given this fossil evidence, modern ammocoetes do not appear to
provide a sound model for the hypothetical ancestral conditions of
vertebrates. Therefore, morphological similarities between ammo-
coetes and invertebrate chordates might be attributed to convergence
or reversal facilitated by similar life habits. For instance, an ammo-
coete has an endostyle—an infrapharyngeal organ that stores iodine
and secretes food-trapping mucous into the branchial system
40
—as
in invertebrate chordates. The ammocoete endostyle later develops
into a follicular thyroid, which is a vertebrate homologue of the chor-
date endostyle
41,42
. The derived state of an ammocoete is seemingly
at odds with this apparent recapitulation, but the ecophysiology of
modern lampreys offers a scenario that is compatible with both. In
the lamprey crown group, the prolonged filter-feeding larval phase
delays the requirement for the endocrine function of a thyroid in
iodine-poor freshwater environments40. This pre-metamorphic state
may have allowed the presumptive thyroid in common ancestors of
the crown-group lampreys to be co-opted as a functional equivalent
of those present in invertebrate chordates. Understanding whether
this co-option event represents convergence or reversal (and whether
it accompanied heterochronic shift) requires a detailed comparison
of the development and determination of the protochordate state of
the endostyle homologue.
Thus, lampreys and cephalochordates remain important branches
for constraining a suite of characters at the crown vertebrate node,
but neither is sufficient for the reconstruction of the ancestral state.
Paradoxically for a vertebrate clade with a notoriously poor-quality
fossil record8,26, here we have robust evidence of a substantial change
in life-history strategy, despite the conservation of adult morphology
of 360million years or more. An ammocoete phase (and the distinct
metamorphosis from filter feeders to predators) evolved somewhere
along the nearly 200-million-year ghost lineage (Fig.4) during which
lamprey habitats broadened to include freshwater. In a clear contrast
to the Palaeozoic stem lampreys
28,36,38
, the early ontogenetic stages of
post-Palaeozoic lampreys (whether fossil or living) occur exclusively in
freshwater environments13,19,24. This implied ecological shift suggests
that ammocoetes were an adaptation to closed, fluctuating fluvial
and lacustrine habitats in which filter feeders are typically less diverse
Haikouella
Myllokunmingia
Haikouichthys
Metaspriggina
Myxineidus
Hardistiella
Mayomyzon
Pipiscius
Priscomyzon
Mesomyzon
Geotria
Mordacia
Lampetra
Lethenteron
Petromyzon
Gilpichthys
Myxinikela
Tethymyxine
R. eos
R. lopheliae
E. burgeri
E. stoutii
E. atami
Myxine
Neomyxine
Euconodonta
Anaspida
Gnathostomata (total group)
Ammocoete-like larval stage
Gametes/hatchlings relatively large
?
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Vertebrata
(crown group)
Cyclostomi
(crown group)
Cyclostomi (total group)
Petromyzontiformes (total group)Myxinoidea (total group)
Date (Ma)
500 400 300 200 100 0
C O S D M Pn P T J K Pg N
Fig. 4 | Time -calibra ted phylogen etic tree of ea rly vertebrat e lineages
shows a late evolut ionary ori gin of a fil ter-feedin g larva. The t ree shows a
portio n of strict con sensus of 360mos t parsimonious t rees based o n 52taxa
and 167morphologi cal characte rs, revised f rom a previous stud y10; the full tree
is shown in Ex tended Data F ig.10. Mean divergenc e estimates for n odes are
also base d on the previous s tudy10. Tips (squares) show the m odern
lamprey-like (blue) an d non-modern-lam prey-like (red) state for the t raits that
are compared i n the right colum ns. Black indic ates missing i nformation. Ti ps
for composit e taxa (bars) show th e chronologic al range of the fossil re cord for
those clad es. Nodes (empt y circle, total no de; filled circ le, crown node) are als o
colour-coded, w ith the ances tral state rec onstruct ed parsimoniou sly and
unambigu ously. Icons asso ciated with ea ch lamprey taxon de note the state o f
larval phase (blue silhouette, ammocoete; red silhouette, non-ammocoete)
and whethe r a circumoral feedi ng apparatus th at consists of p etaliform plate s
and cusps is p resent (compute d tomography ren dering in red). We scor ed
relative size s of gametes or h atchlings in Pipiscius and Priscomyzon on the basis
of the smalle st specimen s describe d in this Articl e (Figs.1p, q, 2a, b), and in
Gilpichthys25 and Hardistiella32 on th e basis of the ser ies of abdomin al
struct ures that were previ ously interpre ted as gonads (Ex tended Dat a
Fig.7i–p). Anaspida wa s scored on the bas is of Euphanerops. Scale at
thebottom sh ows approximate date i n millions of years a go (Ma) along with
period or s ubperiod bo undaries; C, Ca mbrian; O, Ordovician; S, S ilurian;
D, Devonian; M, Mis sissippian; Pn, Pe nnsylvanian; P, Permian; T, Triassic;
J, Jurassic ; K, Cretaceou s; Pg, Palaeogen e; N, Neogene.In th e hagfish c rown
group: E., Eptatretus; R., Rubicundus. E. atami is also known as a sp ecies of the
genus Paramyxine.

412 | Nature | Vol 591 | 18 March 2021
Article
than in marine communities. The presence of such a generalist larval
phase prior to the specialized adult form may have contributed to the
long-term survival of this lineage, after the apparent extinction of their
exclusively non-freshwater relatives.
Online content
Any methods, additional references, Nature Research reporting sum-
maries, source data, extended data, supplementary information,
acknowledgements, peer review information; details of author contri-
butions and competing interests; and statements of data and code avail-
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© The Author(s), under exclusive licence to Springer Nature Limited 2021

Methods
No statistical methods were used to predetermine sample size. The
experiments were not randomized, and investigators were not blinded
to allocation during experiments and outcome assessment.
Anatomical abbreviations
In addition to abbreviations used in Figs.1–3, Extended Data Figs.1–7
use the following abbreviations: af, anal fin; blb, longitudinal bar of
branchial basket; cf, caudal fin; df, dorsal fin; edg, broken edge; gn,
gonads; kd, kidney; kl, kaolinite lump; mo, mouth; my, myomere; ofr,
ridges on oral funnel; os, oral structure; ot, otic capsule; and plp, pos-
terior lateral plate.
Provenance
All specimens of Priscomyzon described in this Article were excavated
and identified by R.W.G., from shale rescued from the roadworks at
Waterloo Farm near Makhanda (also known as Grahamstown) (South
Africa)
43
. All but the smallest specimen (AM5813) were collected from
the same lens as the type specimen (AM5750). AM5813 comes from
an immediately adjacent lens. The specimens are accessioned at the
Albany Museum. The specimens of the Mazon Creek stem lampreys
(Mayomyzon and Pipiscius) have previously been reported from private
collections
25,28
(and transferred to public repositories subsequently) or
were identified by T.M. and M.I.C. in the designated depositories (FMNH
and ROM). The specimens of Hardistiella were previously described31–33,
or reported to T.M. by K. Seymour and D.C. Evans (ROM) and J. Pardo
(University of Calgary).
Anatomical reconstructions
We examined all specimens under a binocular microscope with chang-
ing light angles for the maximum refraction and topographical infor-
mation. In Priscomyzon, the fragility of the specimens precluded any
potentially corrosive methods such as alcohol immersion or ammonium
chloride coating. Their taphonomy includes partial metamorphosis of
the enclosing mudstone during deep burial, during which the tissue
remnants were completely replaced by secondary metamorphic mica,
which converted to kaolinite during uplift. This complete replacement
of the preserved tissues by kaolinite ruled out geochemical analysis
using radiation or spectrometric techniques. The Hardistiella series
comprises three specimens that have previously been described31–33,
and two previously undescribed specimens. Aside from the descrip-
tion of features relevant to the ontogeny of Hardistiella, a complete
description and detailed morphological analyses of the new specimens
are deferred at this time.
Taxonomic identification
Taxonomic identifications are based on several lines of inferences: char-
acters diagnostic to the genus (for example, the numbers of branchial
arches or circumoral elements) and specific to lampreys (for example,
oral funnel); faunal correlates (whether or not any other similar jawless
vertebrates occur in the locality); and fit to the previously known speci-
mens (whether or not the specimen falls into the range of morphological
and taphonomic variations known for the taxon). We assessed each
specimen against modern decay experiments20,44–46 to consider decay
effects on preservation of the soft tissue characters (Extended Data
Fig.8). We then mapped the preserved characters across the specimen
series within each taxon to test for gradual morphological transitions
indicative of ontogenetic sequence, and assigned the specimens to a par-
ticular ontogenetic stage (Extended Data Fig.9). Detailed descriptions
of this ontogenetic analysis are available inSupplementary Information.
Computed tomography scan
The circumoral feeding apparatus of a mature individual of Pipis-
cius (FMNH PF8344) was scanned by K.T. at the Paleo CT Scan
Facility (Department of Organismal Biology and Anatomy, University of
Chicago). Scan parameters used for the part were: voxel size = 11.458µm;
voltage = 65kV; current = 175µA; timing = 2,000ms; filter = 0.5mm Cu.
Scan parameters used for the counterpart were: voxel size = 11.309µm;
voltage = 60kV; current = 180µA; timing = 1,000ms. No distinct layer
of soft tissue was discernible on part or counterpart but the parts did
not fit one another perfectly, which suggests that the body structures
were preserved in thin film (<50µm) at the part–counterpart interface.
Using Mimics (Materialise), K.T. generated an infilling layer over the
surface topography in both part and counterpart (Fig.2e, f). These
negatives show: (a) a mould of the cusps and cast of the petaliform
plates (Fig.2e); and (b) infillings for pulp cavities of the cusps and the
grooves between and within the plates (Fig.2f)
Illustrations
All specimens were illustrated with solid lines to represent topographi-
cal contours and/or changes in mineral compositions, grey shades to
represent organic imprints or tissues, and stipples to show anatomical
structures demarcated by topographical contours.All anatomical
drawings were prepared by T.M.
Phylogenetic analysis
We examined the relationships of the four stem lampreys in a dataset
modified from a previous analysis10. This dataset contains 52taxa and
167morphological characters, and was analysed using PAUP47. Strict
consensus of the 360most parsimonious trees (tree length=344; con-
sistency index=0.544; retention index=0.814; rescaled consistency
index=0.442) is shown in Extended Data Fig.10. Full analytical details
and dataset are available inSupplementary Information.
Reporting summary
Further information on research design is available in theNature
Research Reporting Summary linked to this paper.
Data availability
The digitally reconstructed part and counterpart of Pipiscius (Fig.2e,
f) remain under the copyright of the FMNH, and are available at https://
doi.org/10.6084/m9.figshare.13378628.v1. The original scan data are
property of the FMNH (depository: https://emudata.fieldmuseum.org/)
and are available upon request to the FMNH and the corresponding
author. The cladistic dataset used for our analysis is available asSup-
plementary Information.
43. Gess, R. W. High Latitude Gondwanan Famennian Biodiversity Patterns: Evidence from the
South African Witpoort Formation (Cape Supergroup, Witteberg Group) (Univ. of
Witwatersrand, 2011).
44. Sansom, R. S., Gabbott, S. E. & Purnell, M. A. Decay of vertebrate characters in hagish
and lamprey (Cyclostomata) and the implications for the vertebrate fossil record. Proc. R.
Soc. Lond. B 278, 1150–1157 (2011).
45. Sansom, R. S., Gabbott, S. E. & Purnell, M. A. Non-random decay of chordate characters
causes bias in fossil interpretation. Nature 463, 797–800 (2010).
46. Purnell, M. A. etal. Experimental analysis of soft-tissue fossilization: opening the black
box. Palaeontology 61, 317–323 (2018).
47. Swofford, D. L. PAUP* . (Sinauer, 2017).
48. State of New York Conservation Department. A Biological Survey of the Oswego River
System. Supplemental to Seventeenth Annual Report. (J. B. Lyon, 1928).
Acknowledgements We thank J.-B. Caron, J. Hanken, J. Hurum, P. Janvier, G. Clement,
Z. Johanson, O. Matton, K. Moore, T. Mörss, M. Purnell, S. Gabbott, T. Schossleitner, K. Seymour,
W. Simpson, and S. Walsh for collections access; M. Bronner, A. Chinsammy-Turan, S. Gess,
S. Green, D. Hockman, K. Miyashita, H. Parker, and R. Prevec for logistical support; J. Pardo for
reporting UMPC10210; K. Seymour and D. Evans for reporting ROM78122; and R. Plotnick for
the access to the D. Bardack slide collections. Funding was provided by Chicago Fellows
Program, Vanier Canada Graduate Scholarship, I. W. Killam Memorial Scholarship,
Commonwealth Science Conference Follow-on Grant (T.M.); NSF DEB-1541491 (M.I.C.);
Millennium Trust, South African DSI-NRF Centre of Excellence in Palaeosciences, National
Research Foundation, and the South African Natural Science Collections Facility (R.W.G.).
Author contributions T.M. designed the study, performed morphological and phylogenetic
analyses, prepared igures, and wrote the manuscript; R.W.G. conceptualized the study of

Article
Priscomyzon, performed the ieldwork in the Waterloo Farm lagerstätte, discovered the
specimens of Priscomyzon, and contributed to the morphological analysis of Priscomyzon and
drafting the manuscript; K.T. performed the computed tomography scan of Pipiscius and
provided reconstructions; M.I.C. coordinated the study, identiied the specimens of Pipiscius,
and contributed to drafting the manuscript.
Competing interests The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material available at
https://doi.org/10.1038/s41586-021-03305-9.
Correspondence and requests for materials should be addressed to T.M.
Peer review information Nature thanks Philippe Janvier, Shigeru Kuratani and the other,
anonymous, reviewer(s) for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.

Extende d Data Fig. 1 | Com parison of th e specime ns of P.riniensis to the
same sca le in reverse onto genetic ord er. a, b, An adult (AM5750),
photograph (a) and interpre tive drawing (b). c, d, A juvenile (A M7538),
photograph (c) and interpret ive drawing (d). e, f, A large late larva (A M5816),
photograph (e) and interpret ive drawing (f). g, h, A small late lar va (AM5815),
photograph (g) and int erpretive drawi ng (h). i, j, A small late larva (A M7539),
photograph (i) and inter pretive drawing ( j). k, l, A large early lar va (AM5817),
photograph (k) and inter pretive drawing ( l). m, n, A small early larva (A M5814),
photograph (m) and interpre tive drawing (n). o, p, A hatchling (AM58 13),
photograph (o) and interpret ive drawing (p). Scale bar, 2mm.
q, Reconstr uction of thre e individuals of Priscomyzon, each repre senting a
different o ntogenetic st age. Clockw ise from the ri ght: a hatchlin g carrying a
yolk sac (bas ed on AM5813), tucked in th e meadow of the char ophyte
Octochara c rassa; a juveni le (based on A M7538), attached to the sub strate in
the foregrou nd; and an adult (bas ed on AM5750), looming over the ot her
individua ls and showing it s feeding appara tus. In the back ground, a schoo l of
the coelacanth Selenichthys kowiensis swi m above the charophy te meadow.
Artwor k by K.T.

Article
Extende d Data Fig. 2 | Se e next page for capti on.

Extende d Data Fig. 2 | Adul t and juvenile st ages of P.riniensis. a–g, AM5750.
h–n, AM7538. a, b, Main slab of A M5750. Photograph (a) with angled single-
directio nal light (unlike the ver tical polariz ed light in Fig.1a); and photogr aph
from Fig.1a overlain wit h outlines of the a natomical str uctures iden tified in
this study (b). For interpretive dr awing, see Fig .1b. c, d, Countersl ab of
AM5750. Photogr aph (c) and interpretive d rawing (d). e, Detailed, low-angle-
light photo graph of the oral f unnel, showing th e individual cus ps of the
circumoral fe eding apparatu s and ridges alon g the edge of the oral f unnel.
f, g, Detailed ana tomy of the region s urrounding the l eft (f) and right ( g) eye,
showing eye len ses and otic ca psules. Ow ing to the thickn ess of the pres erved
body, some of the do rsal skeletal st ructures in t he snout (for example, te ctal
cartila ges) cannot be obs erved at the sur face. h, i, Main slab of A M7538.
Photograp h overlain with outli nes of the anatom ical struct ures identif ied in
this study (h) and interpre tive drawing (i). j, Det ailed photogra ph of the
branchial ba sket on main slab. k, De tailed photog raph of the left e ye, showing
the perio cular struct ure. l, Photogra ph of countersla b of AM7538.
m, n, Detailed low-angle-light photographs of the circumoral feeding
apparatus , which consist s of 14grooved, petalifor m plates. m, Mai n slab,
showing ca st. n, Counter slab, showing mould . Scale bar, 5mm (a–d), 2mm
(h, i, l). Definition s of abbreviatio ns are provided in th e legends of Figs .1–3 and
‘Anatomical ab breviations’ i nthe Methods.

Article
Extende d Data Fig. 3 | La te larval sta ges of P.riniensis. a–f, AM5816.
g–k, AM5815. l, m, AM7539. a, b, Main slab of a large late la rva, AM5816
(no counterslab was found). Photograph overlain with outlines of the anatomical
struct ures identif ied in this st udy (a) and interpreti ve drawing (b). c, Detailed
photogra ph of the head, show ing potenti al preserva tion of the pet aliform plates
of the circumo ral feeding appa ratus (arrowhead) a nd additional s oft tissue
struct ures. d, Detailed pho tograph of the br anchial basket a nd digestive tra ct.
e, f, Detailed photo graphs of the p osterior tr unk, with broken li nes showing
partial ou tline of the tail . g, h, Main slab of AM5815, a sm all late larva mi ssing a
large port ion of the snout an d posterior h alf of the trunk . Photograph overla in
with outlin es of the anatom ical struc tures identi fied in this s tudy (g) and
interpret ive drawing (h). i, Photograph of counterslab, which shows posterior
extremit y of the trunk. j, D etailed pho tograph of the h ead, showing ar rangement
of the branch ial arches and po sitions of the s nout struct ures.
k, Detaile d photograph o f the snout and eyes , showing the smal l snout struc tures
and otic cap sules. l, m, Main slab of A M7539, a small late larva (countersl ab has no
anatomic ally informative tr ace). Photograph overla in with outline s of the
anatomic al structure s identif ied in this stud y (l), and interpreti ve drawing (m).
Scale bars , 2mm. Defini tions of abbrev iations are provi ded in the legen ds of
Figs.1–3 and ‘Anatomical abbrevi ations’ in theMe thods.

Extende d Data Fig. 4 | Ea rly larval sta ges of P.riniensis. a–c, AM5817.
d–j, AM5814. a, b, Main slab of AM5817, a large early l arva (no counters lab was
found). Photog raph overlain with ou tlines of the anat omical struc tures
identif ied in this stud y (a) and interpretive dr awing (b). c, Detailed photograph
of the head re gion, showing i ndividual bran chial arches and su rface erosion
struct ures. d, e, Main slab of AM5814, a small ea rly larva. Photo graph overlain
with outlin es of the anatom ical struct ures identif ied in this stud y (d) and
interpret ive drawing (e). f, g, Counterslab. Photograph (f) and interpret ive
drawing ( g), showing individu al branchial arche s. h, i, Detailed photographs of
the oral reg ion in main slab, with ( h) or without (i) broken li nes delineat ing the
circumoral feeding apparatus. j, Detailed ph otographs of th e head region in
countersl ab. Scale bars, 2mm. D efinitio ns of abbreviat ions are provided in t he
legends of Fi gs.1–3 and ‘Anatomical abbrev iations’ in theMet hods.

Article
Extende d Data Fig. 5 | Hat chling sta ge of P.rinien sis, re presente d by
AM5813 car rying a yolk sac . a–c, Main slab. Photog raph (a), photograph
overlain with ou tlines of the ana tomical struc tures identi fied in this s tudy (b)
and interp retive drawing (c). d, e, Detaile d photographs o f the head regio n in
different light angles, highlighting different structures preserved by kaolinite
mass. f, Det ailed photogra ph of the branchial re gion. g, Detailed photograph
of the circumoral feeding apparatus. h–j, Counterslab. Photograph (h),
interpret ive drawing (i) and de tailed photog raph of the head re gion ( j). Scale
bars, 2mm. Def initions of a bbreviation s are provided in the le gends of Figs.1–3
and ‘Anatomical a bbreviation s’ in theMethods .

Extende d Data Fig. 6 | Se e next page for capti on.

Article
Extende d Data Fig. 6 | Vario us growth sta ges of the Mazon C reek stem
lampreys P.zangerli and M.pieckoensis. a–v, Pipiscius (a–h) and Mayomyzon
(i–v) show suites of ch aracters tha t are compatible w ith the Priscomyzon seri es,
including pro minent eyes, an ora l funnel, and shor t branchial bas kets.
a, b, ROM56679, a hatchling carry ing a yolk sac. Phot ograph overlain wi th
outlines of t he anatomical s tructures i dentifie d in this study (a) and
photograph of the counterpart (b). c, d, FMNH PF16082, a hatch ling carry ing a
yolk sac. Phot ograph overlain wi th outlines of th e anatomical st ructures
identif ied in this stud y (c) and photograph of the c ounterpar t (d).
e–h, Comparis on of the specim ens of P.zangerli at t he same scale re veals nearly
identica l suites of morph ological char acters bet ween the hatch ling and adult,
except size and p resence or abs ence of a yolk sac . e, f, Holotype FMNH PF8 346
shown as a phot ograph (e) and interp retive drawing (f), repres enting the
general adul t morpholog y of the taxon and pre served in comp arable
orienta tions to the two h atchling spec imens. g, The la rgest specim en known
for Pipiscius (FMNH PF83 44), showing the circumo ral feeding appar atus in
dorsal vie w. h, The smaller hatch ling (ROM56679), shown in interpre tive
drawing at th e same scale as e–g (two a dult specime ns). i–p, A specimen
referred to M.pieckoensis (FMNH PF8 167), representing a lar va. i–l, Main part .
Photograp h (i), photograph overlain w ith outlines of t he anatomical s tructures
identif ied in this stud y (j), inter pretive drawing ( k), and scanned phot ograph
taken in the 1960 s by D.Bardack to show the ori ginal state of t issue
preser vation in this spe cimen (l). The pho tograph in l was di scovered in the
slide collec tions of D.Bardack . A full view of the sno ut can be seen i n figure 2 of
ref. 28. The sp ecimen was da maged in the snout, a nd the thin film o f organic
tissues h as deteriora ted across the e ntire specime n. m–p, Detailed
photogra phs and illustrat ion of the head re gion. Photog raph in low-angle
lighting to r eveal surface tex tures (m), interpretive dr awing (n), photograph in
high-angle li ghting to reveal th e film of prese rved tissue s (o) and photograph
of counterpart in high-angle lighting for comparison (p). q, Scan of photogra ph
taken in the 1960 s by D.Bardack, to show orig inal state of ti ssue preser vation in
the head re gion of FMNH PF568 7, a juvenile of M.pieckoensis. The pho tograph
in q was discovered i n the slide collec tion of D. Bardack . Further detail s can be
seen in f igures 2–4 of ref. 28. r–v, Comparison of t he specimen s of M.pieckoensis
at the same s cale reveals chara cter transit ions across on togeny, including the
decreasi ng relative propor tions of branc hial region. r, s, Holoty pe FMNH
PF5687 shown a s a photograph (r) and int erpretive drawi ng (s), representing
the juvenile st age. t, Interp retive drawing of FM NH PF8167 at the same sc ale as
r, s (juvenile), u, v (adult). u, v, The largest know n specimen (RO M56787) shown
as a photogr aph (u) and interpreti ve drawing (v), representi ng the adult sta ge.
Scale bars , 2mm (a–d, i–l, m–p), 5mm (e–h, r–v). Definiti ons of abbreviat ions
are provided in t he legends of Fig s.1–3 and ‘Anatomic al abbreviatio ns’ inthe
Methods.

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Article
Extende d Data Fig. 7 | Grow th series o f H.montanensis. This seri es shows
suites of ch aracters tha t are compatible w ith those of othe r Palaeozoic ste m
lampreys, incl uding prominent e yes, an oral funnel, a nd short branc hial
baskets. T his series a lso document s gradual chara cter transit ions in the
decreasi ng body depth, in creasing inte rocular dista nce and progres sively
slender prof ile of the oral fu nnel. Compar ison reveals size di screpancie s across
the juvenile–ad ult transitio n and between t he sexually mature , gravid
specimen and other adults (discussed in section A-3a of the Supplementary
Informatio n in compariso n to modern lamprey s). a–l, Hardistiella
montanensis. a, b, Photograph (a) and i nterpretive draw ing (b) of late larva l
stage repre sented by CM4505, sho wing the deep er body than in any la ter
ontogenetic stages. c, d, Juvenile stage, repr esented by ROM781 22, showing
intermediate body depth. Photograph (c) and interpretive draw ing (d).
e–l, Adult stage re presented by CM63 079 (e, f), UMPC10210 (g, h) and
UMPC7696 (i–l). e, f, CM63079—incomplete, b ut potentially t he largest
specime n—shown as a photog raph (e) and interpret ive drawing (f).
g, h, UMPC10210, showing a n advanced sta ge of decay relative to o ther
specime ns, shown as a photo graph (g) and in terpretive draw ing (h).
i–l, UMPC7696 associa ted with pote ntial gonads (prob ably represen ting a
gravid femal e with ovaries or e ggs), shown as a photog raph (i) and inter pretive
drawing (l). k, De tailed photog raph of the abdo minal region of th e main part,
showing the c ircular struc tures interpre ted in ref. 32 as gonads . l, Tracing of the
anatomic al structure s in k (in grey). m–p, The abdomina l structure s
wereidentif ied as gonads in t he stem myxino id Gilpichthysgreenei from th e
Mazon Creek fa una, indicati ng a small number of la rge ovaries or eg gs (white
arrowhead s) in this taxon. The se examples, wh ich are shown here in v arious
forms of develop ment and stat es of preserv ation, bolste r the gonadal
interpret ation for the ser ial, circular abdo minal struct ures in the holot ype of
the stem lamp rey Hardistiella UMPC7696 (k, l). In Gilpichthys, the stru ctures
appear to deve lop in a small number (3–5) of i ron concretion s as in ROM56389
(m), or paired serie s of fewer than a dozen circu lar depression s as in FMNH
PF234 64 (n). o, p, FMNH PF8464 shows a s eemingly ma ture state of
developmen t: 11circular stru ctures of iron-ri ch recryst allization, imp lying that
the conten ts were encaps ulated and chem ically distinc t from the rest of t he
body. The numb er agrees wi th the 11 prese nt in FMNH PF23 464, shown he re as a
photogra ph with low-angle lig hting (o) and as a detail ed photograph o f the
gonads in hig h-angle, high-ex posure lightin g (p). Scale bars, 5mm. Def initions
of abbreviat ions are provide d in the legends of Fi gs.1–3 and ‘Anatomical
abbreviat ions’ in theMetho ds.

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Article
Extende d Data Fig. 8 | Com parison of a natomical t raits prese rved in the
specim ens of Palaeo zoic stem lamp rey describ ed in this Ar ticle. a, Moder n
decay ser ies of ammoco etes and adult lam preys, after a prev ious publicat ion20.
Codes for the se reference de cay series are a s follows. Yellow, pristine; oran ge,
decayin g; red, onset of lo ss; terminal po int, complete lo ss. The x axis s hows
number of days , whereas yaxis ran ks decay sta ges. For each fossi l specimen
(b–p), bars shown in orig inal colours (ye llow, orange, and red) repr esent
charact ers preserve d in the specime n; 50% transparen t (brown) bars repre sent
charact ers preserve d in the specime n but in a different for m than in the
modern refe rence; grey bars re present chara cters missi ng in the specime n; no
bars shown de notes that, in pr inciple, chara cters canno t be assesse d in the
specime n (for example, ‘slanting g ill openings’ c annot be deter mined in
dorsoventr ally compresse d specimens , whereas ‘gill sy mmetry ’ cannot be
assess ed in transvers ally compress ed specimen s). b–i, Priscomyzon riniensis.
b, AM5750, represen ting the adult st age. c, AM7538, repres enting the juve nile
stage. d–f, AM5816 (d), AM5815 (e), and AM7539 (f), all representing th e late
larval stage. g, h, AM5817 ( g) and AM5814 (h), represen ting the early lar val
stage. i, AM5813, repres enting the hatc hling stage. j, k, Pipisc ius zangerli. ROM
5667 9 ( j) and FMNH PF160 82 (k), both represe nting the hatc hling stage.
l, Mayomyzon pieckoensis. FMNH PF8167, represen ting the late lar val stage.
m–q, Hardistiella montanensis. m, CM4505, repr esenting the l ate larval sta ge.
n, ROM78122 , representin g the juvenile sta ge. o–q, CM63079 (o), UMPC10210
(p), and UMPC7696 (q), all representing t he adult stage. I nterpretive dr awings
in b–q are not to scale .

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Article
Extende d Data Fig. 9 | De terminati on of ontogen etic stage s for the
Palaeozoic stemlamprey specimens. This comparison reveals gradual
transiti ons of charact ers in each taxon , allowing the de signation of
ontogene tic stages and c orroboratin g their taxonom ic identity. Each
ontogene tic series of s tem lamprey shows an e arly expression of c haracter
states t hat, in modern lam preys, develop only in th e adult phase (dark blue
shades in c–f), conf irming the ab sence of the am mocoete pha se in these ste m
taxa. a, Modern lampreys. Reference ontogenetic sequence and comparison of
ontogene tically variabl e characters t hat are also prese rved in the stem l amprey
specime ns, indicated i n blue shades. I mages are after a p revious public ation48
(adult, P. marinus) or courte sy of G. Kovalchuk (lat e larva and juvenile ,
Entosphenus tridentatus), except for the hat chling and early l arva (photogr aphs
by T.M., P. marinus). b, Size variations am ong the specim ens of P.riniensis.
Principa l component1 is us ed as a composit e size metric tha t correlates w ith
ontogenetic stages (xaxis), whereas the spec imens are similar in b ody length
from larval t o juvenile stage s (yaxis). For metho dological de tails, see the ‘A-3a.
Size variat ions’ secti on oftheSupplement ary Informati on. For original
measurem ents and princ ipal compone nt scores, see S upplementa ry Tables3
and 4, respec tively. c–f, Ontogenet ic series of four Pa laeozoic stem lam preys:
P.riniensis (c); P.zangerli (d); M.pieckoensis (e); and H.montanensis (f). In
addition to t he character s that vary in mod ern lamprey ontoge ny (blue shades),
the table co mpares charac ters that var y ontogenetic ally in Priscomyzon (re d
shades) and in Hardistiella and Mayomyzon (gree n shades). Each row
represen ts a characte r; the lighter sha de shows the immatu re state (descri bed
in the left most column) and the d arker shade shows the m ature state
(describe d in the rightmo st column). Interm ediate shades i ndicate
polymor phism within th e stage (absence a nd presence mi xed) or interme diate
state (charac ter descri bes a continuu m). An empty box wit h a question mark
denotes m issing informat ion. Each sta ge represente d in the fossil record i s
linked to inter pretive drawing s of the specime ns (represent ative specime ns for
nonlarval s tages of Mayomyzon and Pipiscius). For detai led information a nd
discussi on of the charac ters, see the ‘A-3f. Defini tions of ontoge netic
charact ers’ sectio n oftheSupplementa ry Informatio n.

Extende d Data Fig. 10 | Phylo genetic tre e of early verte brate linea ges
resulti ng from our ana lysis, showi ng the relatio nships of st em lampreys
and other cyclostomes. The tree shows str ict consen sus of 360most
parsimoni ous trees bas ed on 52taxa and 167morpholo gical charac ters, revise d
from a previou s publication 10. Topology is similar to th e previously pre sented
consensus tree10, but differs i n: the deeply ne sted, stem-pe tromyzontiform
position o f Pipiscius; the stem-myx inoid aff inity of Gilpichthys; th e position of
Myxineidus as outg roup to all the rest of p etromyzonti forms; and the collap se
of crown node s into polyto mies for both myx inoids and petr omyzontiforms .
Filled circle s indicate crown n odes, and empt y circles show tot al nodes or the
most inclus ive nodes of enti rely extinct lin eages. Black , nonvertebra te
outgroup s; green, stem ver tebrates; ma genta, stem cycl ostomes; purp le,
anaspids (ste m cyclostomes); red, my xinoids (hag fishes); oran ge,
petromyz ontiforms (lamp reys); blue, gnathostom es (total group). For the
methodol ogy and analy tical detai ls, see the ‘Par t B’ secti on
oftheSupplementary Information.