The rise of predation in Jurassic lampreys

Domain: earth_science
Published: 2023-10-31
Editorial outcome: minor_revision

Abstract
Lampreys, one of two living lineages of jawless vertebrates, are always intriguing for their feeding behavior via the toothed suctorial disc and lifecycle comprising the ammocoete, metamorphic, and adult stages. However, they left a meager fossil record, and their evolutionary history remains elusive. Here we report two superbly preserved large lampreys from the Middle-Late Jurassic Yanliao Biota of North China and update the interpretations of the evolution of the feeding apparatus, the lifecycle, and the historic biogeography of the group. These fossil lampreys’ extensively toothed feeding apparatus differs radically from that of their Paleozoic kin but surprisingly resembles the Southern Hemisphere pouched lamprey, which foreshadows an ancestral flesh-eating habit for modern lampreys. Based on the revised petromyzontiform timetree, we argued that modern lampreys’ three-staged lifecycle might not be established until the Jurassic when they evolved enhanced feeding structures, increased body size and encountered more penetrable host groups. Our study also places modern lampreys’ origin in the Southern Hemisphere of the Late Cretaceous, followed by an early Cenozoic anti-tropical disjunction in distribution, hence challenging the conventional wisdom of their biogeographical pattern arising from a post-Cretaceous origin in the Northern Hemisphere or the Pangean fragmentation in the Early Mesozoic.

Introduction
As a lineage of the living jawless vertebrates, lampreys have great weight in the study of vertebrate evolution1–5. They are characterized by their peculiar feeding behavior of eating blood or cutting off tissues from the hosts or prey to which they firmly attach via their toothed oral sucker6,7. In such a way, lampreys play a significant role in the aquatic ecosystem and, in some cases, where they are non-native, even bring tremendous loss to the fishery economy4. These jawless fishes have been in existence for ca. 360 million years but left an extremely patchy fossil record in the post-Carboniferous period, with only two species known from the Cretaceous2,3,8,9. Despite the superficially conservative morphology throughout their history, from the simply assembled teeth in Paleozoic fossils, lampreys’ feeding apparatus, especially the size, shape and arrangement of the keratinous teeth, was substantially reformed and enhanced to the pattern of modern species2,3,6,7,9–12. And evidently, departing from their Paleozoic kin with non-ammocoete larvae and expanding the habitats to the freshwater domain, lampreys changed the life-history strategy sometime before the Cretaceous by evolving the ammocoete and metamorphic stages3,9,13–15. Eventually, they established their current diversity and anti-tropical distribution5,16. However, these scenarios were poorly understood due to the lack of reliable fossil record and the disputed lamprey phylogeny, especially that of the crown-group lineages1–5,16. Here we shed light on these issues by reporting two lampreys from the Jurassic terrestrial fossil Lagerstätte Yanliao Biota of North China17. These fossil lampreys were exquisitely preserved with a complete suite of feeding structures, including the well-developed movable biting plates on the tongue-like piston, which has never been clearly recognized in previously known fossil lampreys and astonishingly resembles the pouched lamprey (Geotria australis12) now confined to the Southern Hemisphere. Bridging the recorded fossil and extant lampreys, these fossils offer an opportunity to reconstruct the evolutionary process and the ancestral state of modern lampreys’ feeding biology. As key constraints of the evolutionary timeline of its group, they also make it possible to assess the coevolutionary interactions with the potential hosts (or prey) and their implications for the establishment of the modern-type life-history mode. Also, based on the revised lamprey phylogeny, the early biogeographical history of modern lampreys was reconstructed and their ‘poles-apart’ distribution pattern was reinterpreted.

Results
Systematic paleontology
Order. Petromyzontiformes Berg, 194018
Genus Yanliaomyzon gen. nov.
Typespecies. Yanliaomyzon occisor gen. et sp. nov.
Diagnosis. Stem lampreys with oral discs well-toothed in anterior and lateral fields; anterior and lateral oral disc teeth closely arranged, dorsally truncated, spatulate in shape with the slightly concaved undersurface of the free edge protruding a shallow blade; posterior disc teeth lacking, anterior and lateral circumoral teeth elongate and trihedral in shape; supraoral lamina large and consisting of two stout central cusps flanked by wing-like lateral extensions; transverse lingual lamina very large with the apices of three cusps interlocking with the supraoral lamina in vivo.
Included species. Yanliaomyzon ingensdentes gen. et sp. nov.
Etymology. ‘Yanliao’ derives from Yanliao Biota, a Jurassic terrestrial Lagerstätte from North China17, where these fossils were discovered; ‘myzon’ (Greek), sucker.
Yanliaomyzon occisor gen. et sp. nov. (Figs. 1a–e, 2e, f, Supplementary Figs. 1g–i, 2a–g, k, l)
Etymology. Latin ‘occisor’, meaning ‘killer’, refers to the powerful hunting skill of the species.
Holotype. IVPP V15830, a completely preserved lamprey.
Paratype. IVPP V18956A, B, a lamprey with the head and anterior trunk preserved.
Horizon and locality. Tiaojishan Formation, Oxfordian, earliest Late Jurassic, ca. 158.58–160 million years ago (Ma)19; Daxishan, Toudaoyingzi Town Jianchang County, Liaoning Province (Holotype), and Nanshimen Village, Gangou Town, Qinglong County, Hebei Province (Paratype), China.
Diagnosis. The supraoral lamina spanning completely the lateral rims of the oral aperture, with the central cusps flanked immediately by two smaller projections; 16 circumoral teeth; the tail region occupying slightly less than 28% of the total body length.
Yanliaomyzon ingensdentes gen. et sp. nov. (Figs. 1f–h, 2a–d, e, Supplementary Figs. 1a–f, 2h–j)
Etymology. Latin ‘ingens + dentes’, meaning large teeth, refer to the large cuspid laminae on the gouging piston.
Holotype. IVPP V16715A, B, a completely preserved individual.
Paratype. IVPP V16716A, B, an exquisitely preserved toothed oral disc and the laminae on the tongue-like piston.
Horizon and locality. Daohugou beds, Callovian, late Middle Jurassic, ca. 163 Ma19 in Wubaiding Village, Reshuitang County, Liaoning Province, China.
Diagnosis. The supraoral lamina occupying roughly one-third of the rim of the oral aperture; the transverse lingual lamina almost equaling the supraoral lamina in width; ca. 23 circumoral teeth; the tail region occupying slightly more than 40% of the total body length.
Description and comparison
Yanliaomyzon occisor and Y. ingensdentes are fairly large (Supplementary Table 1) with the former one (642 mm) being among the largest of the group, only smaller than the anadromous sea lamprey Petromyzon marinus (maximum adult length 1200 mm, same for below), Pacific lamprey Entosphenus tridentatus (850 mm), pouched lamprey Geotria australis (788 mm) and Arctic lamprey Lethenteron camtschaticum (790 mm)7,12.
The most conspicuous features of Yanliaomyzon species are the extensively toothed oral disc (od) and tongue-like piston (pt) (Supplementary Note 1, Figs. 1, 2a–f, Supplementary Figs. 1b–d, h, i, 2h–l). Their dentitional arrangement, similar to one another in general morphology, strikingly resembles that of pouched lamprey Geotria australis (Fig. 2g) currently distributed in the Southern Hemisphere10,11. The oral disc occupies 5.2% of the total body length in Y. occisor, which falls within the range (2.7–6.3%) of the living flesh-eating lampreys11. The size of the oral disc cannot be accurately measured for Y. ingensdentes because this part was distorted during the preservation. In the anterior and lateral fields of the oral disc, the closely arranged disc teeth (dt) are dorsally flattened, slightly concave in the posterior surface with the blade-like dorsal edge pointing toward the center of the oral aperture. The morphology of these teeth, together with their absence in the posterior field, is reminiscent of the pattern in pouched lamprey Geotria australis10,11,20, although they are less numerous than in Geotria20. The circumoral teeth (cot) of Yanliaomyzon occisor and Y. ingensdentes are similar in general morphology but those in the former are proportionally larger and less numerous than in the latter. These teeth are much larger than other oral disc teeth and trihedral in shape, with the longest of the three cutting edges facing slightly sideways and posteriorly (Fig. 2a–f). By analogy, with such an arrangement, these oral disc teeth might function similarly to their counterparts of Geotria australis in allowing the forward sliding of the oral disc over the prey’s body surface and resisting any tendency to slip backward or laterally of the disc, thereby coordinating the biting movement of the transverse lingual lamina (tl) on the tongue-like piston cartilage10,11. The posterior circumoral teeth are lacking in Yanliaomyzon species as in Lampetra species11,12 and Geotria macrostoma19, whereas they are usually the largest teeth in the complete circumoral series in Geotria australis10,20. The supraoral laminae (so) are very large and all have a pair of sharp central cusps flanked by a pair of large and curved wings as in Geotria10,20. However, this lamina of Yanliaomyzon occisor differs from that of Y. ingendentes in its longer lateral extension and two accessory projections lateral to the central cusps (Figs. 1b, f, g, 2a–d, f, Supplementary Information). The wide infraoral laminae (io) of both species are surmounted by a straight row of about nine to ten stout cusps. The transverse lingual laminae are remarkably large and occupy most of the part of the oral aperture (Fig. 2a–f, Supplementary Figs. 1g–i, 2h–j). This lamina is thus proportionally much larger than its counterpart in living lampreys10–12,20, whereas this structure is likely much less developed, if present, in all other known fossil lampreys3,8,9,21–27. Despite the variations in the relative size and the shape of the ventral part, the general morphology of this key feeding structure is similar in both Yanliaomyzon species. This toothed lamina is convexly curved and characterized by a prominent central cusp flanked by two slightly smaller lateral cusps like in the feeding adults of Geotria australis10,11. The tips of these three cusps, when raised upward in life, interdigitate with the grooves on the rear side of the supraoral lamina (Fig. 2a–f), which, again, recalls the feature unique to Geotria australis among living lampreys and allows powerful biting and removing large amounts of flesh from the prey10,11. The cusps of the longitudinal lingual laminae (ll, ll.l, ll.r) are strong and closely arranged, making this lamina an extensively serrated cutting edge (Figs. 1b, 2a–d).
The prebranchial region is longer than the branchial apparatus (ba) (Fig. 1c–e, f, h), of which the range can be roughly determined by the positions of the branchial pouches (bp) and external gill openings (go). The eyes (e) are medium-sized and oval in shape, with the otic capsules (oc) positioned immediately behind (Fig. 1c, d, f). The gular pouch (gp) of these lampreys (Figs. 1b, e, 2a–d, f, Supplementary Fig. 2h–l) is small and seemingly has two lumina (Figs. 1b, 2a–d, f). This structure was also observed in the Cretaceous lamprey Mesomyzon mengae9 but never seen in any other fossil lampreys3,8,21–27. Where present, this structure in pre-spawning and spawning males may have the function of courtship display or energy storage for spawning migration for anadromous species28. Among living lampreys, seven out of the 12 species with gular pouch are anadromous12,20,28.
The dorsal fin (adf) of Yanliaomyzon is fairly long and extends anteriorly until the level of the fourth gill pouch (Fig. 1c–e, f, h) as in Mesomyzon mengae9. Just since the position of the cloaca (ca), a long precloacal skin fold (paf) is developed in both species and extends anteriorly to the anterior branchial region (Fig. 1c–e, f, h).
In the position of the intestinal tract, several tooth-bearing jaw bones and possibly skull bones of some unidentified bony fishes and some skeletal relics (ic) are preserved in Y. occisor (Supplementary Fig. 2a, c–f) and Y. ingensdentes (Supplementary Fig. 1a, f), respectively. Together with the anatomy of the feeding apparatus described above, the bones and skeletal relics point to a flesh-eating habit for these fossil lampreys, making them the oldest record of its group with feeding mode clearly specified so far.
Phylogenetic analyses
Calibrated by these Jurassic stem lampreys and based on an updated character data matrix (Supplementary Note 2), the Petromyzontiform timetree is revised and contrasts with those in other relevant studies3,16,29–34, especially in the age and phylogeny of the crown-group lampreys. The all-compatible consensus tree shows that within the cyclostome monophyly and crownward to the basal Paleozoic lampreys, three Mesozoic species, Mesomyzon mengae, Yanliaomyzon ingensdentes and Y. occisor are resolved successively along the stem, with the latter two immediately basal to crown-group lampreys (Fig. 3). This is different from a recent study where Mesomyzon mengae is placed in the crown-group lampreys as the sister to Petromyzonidae34. In current crown-group interrelationships, Geotria (Geotriidae) was resolved as the earliest diverging lineage, sister to the pair of Mordacia (Mordaciidae) and Petromyzontidae, whereas other studies either support Geotriidae-Mordaciidae29,34 or Geotriidae-Petromyzontidae pairs16,32,33, or their interrelationships are unresolved in a basal polytomy3,31. Mesomyzon, the earliest lamprey with a modern triphasic life cycle known so far3,9,13, diverged from other lampreys in 181.8 (236.0–163.00) Ma (median age and 95% HPD interval, same for below). The petromyzontiform crown was estimated to originate in 78.0 (122.7–40.8) Ma, which is roughly 90 million years (Myr) younger than the recent total-evidence inference (169.82 Ma)29 and roughly 240–97 Myr younger than previous molecular-dating estimate (280–220 Ma)30, respectively. Our estimation of the crown age is also ca. 140-95 Myr younger than those (171.59–219.29 Ma) in a recently reconstructed lamprey phylogeny34. Within the crown, the split of Southern and Northern Hemisphere lampreys was estimated to occur in 58.4 (95.0–29.5) Ma, which is again much younger than the aforementioned estimations29,30,34. The inferred phylogeny of Petromyzontidae is similar to the one presented by Brownstein and Near34 but the age (ca. 26 Ma) of the most recent common ancestor (MRCA) of this family is again much younger than 94.1 (139.56–62.41) Ma in that study34, with the appearance of most living Northern Hemisphere lamprey lineages dated to after 10 Ma, still younger than the estimation (ca. 20 Ma) of the same study34.
The evolutionary rates of lampreys’ feeding apparatus
Regarding the evolutionary rates of the feeding apparatus, the Mesozoic species act as the ‘watershed’ of the history of lampreys (Supplementary Discussion). The relative rates along the branches downward and crownward to themselves greatly differ, with the former almost ten times the latter (Supplementary Fig. 3). The evolutionary rate is rather low along the stem above Yanliaomyzon in the Late Jurassic, and then the rates are very uneven after the emergence of the MRCA of the crown-group lampreys, with the rate at the branch subtending Mordaciidae and Petromyzontidae ca. 77 times that low rate toward Geotriidae (Fig. 3). This evolutionary rate heterogeneity reflects the novelties of the feeding apparatus in the Mesozoic lampreys relative to the Paleozoic species and highlights the marked reinforcement of those Geotria-like biting structures in Yanliaomyzon species, which approximate the state of the MRCA of the crown-group lampreys. It also signifies the rapid diversification of the feeding habits during the early history of the living lamprey lineages (Fig. 3).
The reconstruction of the ancestral feeding mode of living lampreys
Based on the characteristic reconstruction of the feeding mechanism (Supplementary Codes 4–6), the MRCA of all living lampreys probably had a feeding apparatus comprising spatulate oral disc teeth, a large supraoral lamina with two central cusps flanked by lateral flanges, a U-shaped movable transverse lingual lamina with three large cusps, and a parenthesis-shaped longitudinal lingual lamina. In general, the assembly of such a feeding apparatus is fairly similar to those in the feeding adults of pouched lamprey Geotria australis10,11 and Yanliaomyzon species described here and points to a flesh-feeding habit10,11 (Fig. 3).
The ancestral distribution reconstruction
Reconstructions of ancestral areas were based on the assignments of fossil and extant lineages into nine geographic provinces (Fig. 4) (Methods and Supplementary Codes 7, 8) by incorporating the scheme proposed in a recent study34. The MRCAs of the Cretaceous (Mesomyzon mengae) and Jurassic (Yanliaomyzon species) plus the crown-group lampreys were all estimated to originate most likely in Eastern Laurasian landmass, with the MRCA shared by Mesomyzon having an Early Jurassic age and that shared by Yanliaomyzon a late Middle to early Late Jurassic age (Fig. 4). The MRCA of all living lampreys probably originated in the Southern Hemisphere (southern Australia plus New Zealand + South America, with a combined probability of ca. 40%) of the Late Cretaceous (ca. 78. Ma, middle Campanian). After the divergence of Geotria, the MRCA of Mordacia + Petromyzontidae (Northern Hemisphere lineages) was most likely (with a probability of ca. 40 %) also a Southern Hemisphere species with an age of early Cenozoic (ca. 58 Ma). The MRCA of the Northern Hemisphere lampreys (Petromyzontidae) was estimated to arise in Western North America (Northeastern Pacific) of the late Oligocene (ca. 26 Ma), followed by the divergence of two main lineages that began to diversify during the middle Miocene in Western North America (ca. 15 Ma) and in Eastern North America or Europe (Northern Atlantic) (ca. 13 Ma), respectively.

Methods
Fossil specimens
This research complies with all relevant ethical regulations. This study is based on two specimens of Yanliaomyzon occisor gen. et sp. nov. and two specimens of Yanliaomyzon ingensdentes gen. et sp. nov., which are housed at the Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences. The specimens of Yanliaomyzon occisor gen. et sp. nov., IVPP V15830 comes from the Tiaojishan Formation, earliest Late Jurassic, ca. 158.58–160 Ma19 of Daxishan, Toudaoyingzi Town Jianchang County, Liaoning Province, and IVPP V 18956A, B from the corresponding layers of Nanshimen Village, Gangou Town, Qinglong County, Hebei Province, China. The specimens (IVPP V16715A, B, IVPP V 16716A, B) of Yanliaomyzon ingensdentes gen. et sp. nov. come from the Daohugou beds, late Middle Jurassic, ca. 163 Ma19 in Wubaiding Village, Reshuitang County, Liaoning Province, China. The materials were collected by Xiaoling Wang and Min Wang of the Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences.

Data
Different from the recent comprehensive vertebrate and cyclostome phylogeny29, this study focuses on resolving the systematic positions of the Jurassic fossil lampreys within the petromyzontiform as well as their closely related taxa, thus the remote taxa were not included, e.g., the hemichordate (Saccoglossus kowalevskii), tunicate (Ciona intestinalis), cephalochordate (Branchiostoma floridae), Pikaia, controversial Cambrian vertebrates (Haikouella, Haikouichthys, Metaspriggina, and Myllokunmingia), and the lineage leading to the gnathostomes (jawed vertebrates). In this context, the dataset includes 25 extant taxa and 20 fossils from Cyclostomi (total group) and combines both morphological characters and molecular sequences, with morphological characters for all taxa (208 characters, see Supplementary Note 2), 16S gene for 10 extant species (772 sites) and CO1 gene for 11 extant species (721 sites). The character description and revision are listed in Supplementary Note 2.
The Bayesian total-evidence dating analysis was performed using the software MrBayes 3.2.864. This version was not formally released when performing this study and the executable was compiled from the latest source code (https://github.com/NBISweden/MrBayes). We describe the models, priors and Markov chain Monte Carlo (MCMC) settings for the analysis. The data matrix and MrBayes commands are given in Supplementary Codes 1 and 2.

Substitution models
The Mkv model65 was used for the morphological data correcting for the variable coding bias (only variable characters are coded in the matrix). For each gene, the HKY model66 was used with an independent transition-transversion rate ratio (κ) and exchangeability rates. Rate variation across characters (sites) was modeled using the discrete gamma model67, while rate variation across data partitions was drawn from a uniform Dirichlet distribution68 under the constraint of the average rate across characters (sites) being 1.0.
We used the default priors for the substitution parameters, e.g., Exponential (1.0) for the gamma shape parameter (α), Beta (1, 1) for the transformed transition-transversion rate ratio (κ/(1+κ)), and a uniform Dirichlet distribution for the exchangeability rates (π).

Total-evidence dating
Traditional phylogenetic analyses infer branch lengths measured by expected changes per character, which are products of geological times (myr: million years) and evolutionary rates (expected changes per character per myr). Without the fossil ages and clock-model assumptions, the two components are confounded and indistinguishable. In the total-evidence dating analysis, we use fossil ages as tip calibrations to co-estimate divergence times and evolutionary rates. To investigate evolutionary rate heterogeneity among separate morphological regions and between morphological characters and molecular sequences, the morphological characters were divided into three partitions, that is, feeding mechanism (51 characters), branchial apparatus (22 characters) and others (135 characters) (Supplementary Code 2). Eventually, each of the three morphological partitions had an independent clock model, and the two gene partitions share another independent clock model.
All partitions share the base clock rate (c), which was assigned a lognormal (−6, 1.0) prior with mean 0.004 and standard deviation 0.005. The evolutionary rate variation was modeled by the relative rate (r) multiplied by the base rate (c). The relative rates in each of the four relaxed clocks follow independent and identically distributed (i.i.d.) lognormal distributions69 with mean 1.0, and the variance of each clock (σ) was assigned an Exponential (1.0) prior by default.
Apart from the clock model, the other component is the timetree. The timetree was modeled by the fossilized birth-death process68,70,71 with speciation rate λ, extinction rate μ, fossil-sampling rate ψ, extant-sampling proportion ρ, and conditioned on the root age (tmrca). The ages of the fossils were fixed to their geological times (see Supplementary Code 2). For the priors, tmrca was assigned an offset-exponential distribution with mean 600 and minimum 500 Ma referring to the oldest fossils, d = λ–μ ~ Exponential (100) with mean 0.01, v = μ/λ ~ Uniform (0, 1) and s = ψ/(μ+ψ) ~ Uniform (0, 1). The extant-sampling proportion was fixed to 0.3, assuming 25 out of about 80 living species were sampled and included in the analyses.
Two independent MCMC runs with 4 chains per run (1 cold, 3 heated) were run 30 million generations and sampled every 400 generations. The first 25% samples were discarded as burn-in and the remaining were combined after examining the convergence of both runs. The posterior tree samples were summarized as an all-compatible consensus tree (Supplementary Code 3), which displays all compatible clades in the posterior trees as bifurcations, including those with a probability smaller than 0.5.

Ancestral state reconstruction
To study the evolution of lampreys’ feeding modes, we inferred the ancestral states of the feeding mechanism characters for the node of the most recent common ancestor (MRCA) of the living lampreys (crown group). This inference was done in the same total-evidence dating framework described in the previous section, except for additionally constraining the crown group as a monophyletic clade (Supplementary Codes 4–6).
We also reconstructed the ancestral geographical ranges for several key nodes, including that of the MRCA of crown lampreys (Fig. 4). The geographic allocations of the living lampreys were coded into nine provinces (Fig. 4) (Methods and Supplementary Codes 7, 8) in addition to the scheme proposed by a recent study on the phylogeny and historic biogeography of modern lampreys34. In our analysis, the geographical distributions of some living species were revised from the assignments in ref. 34: Caspiomyzon wagneri was coded as a European taxon rather than an Asian one34 based on their geographical isolation from the distribution of the species in temperate Asia4,12 and the European distribution of the congeneric species34; Entosphenus tridentatus was coded to be distributed in both Western North America and Asia because it is also distributed in Japan12, so was Lethenteron camtschaticum12. We used the subtree of the stem and crown lampreys (rooted at Priscomyzon) inferred from the total-evidence dating analysis and fixed the node ages to their median estimates.

CT scanning and computerized tomography
X-ray micro-computerized tomography was adopted for scanning the oral disc and the dentition of IVPP V15830. The scanning was carried out using the 225kV micro-computerized tomography (developed by the Institute of High Energy Physics, Chinese Academy of Sciences (CAS)) at the Key Laboratory of Vertebrate Evolution and Human Origins, CAS72. The specimen was scanned with a beam energy of 140kV and a flux of 120μA at a resolution of 36.07μm per pixel using a 360° rotation with a step size of 0.5°. A total of 720 projections were reconstructed in a 2048*2048 matrix of 1536 slices using a two-dimensional reconstruction software developed by the Institute of High Energy Physics, CAS. The data then was analyzed by using VGstudio 2.2 to produce the 3D images (Supplementary Fig. 2l).

Illustrations
All drawings of Figs. 1 and 2 were done with pencils by the author. To distinguish the types of dentition and the associated structures of the feeding mechanism, different colors were filled in corresponding regions using Adobe Photoshop CS6. The phylogenetic trees were visualized in FigTree (v1.4.4) and adjusted in Adobe Illustrator CS6 and Affinity Designer (v1.8.4). The digital reconstructions and models (Figs. 1e, h, 2d, f) were done by NICE Vistudio (Paleovislab, IVPP).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an outstanding contribution to our knowledge of lamprey phylogeny and biogeography through\nthe integration of all known fossil and virtually all extant lampreys (Geotria macrostoma excluded) that feed post-metamorphosis. The discovery of two new fossil lampreys has enabled this\nbreakthrough.\nI have a number of comments and suggested changes to improve the manuscript.\nThe authors refer to the Jurassic era, but shouldn’t it be Jurassic Period?\nIn the Abstract, the three phases of the life cycle should be specified for clarification.\nThe word “hosts” at the end of the Abstract and Introduction should be replaced by prey because,\ngiven their oral and lingual dentitions, the two newly-described fossil lamprey species were more\nlikely predators rather than parasites.\nIn the Abstract, the word “the” should precede Pangean supercontinent.\nIn the Introduction, it is unclear to me what is meant by “simply assembled analogue”. This needs\nto be clarified.\nIn the Introduction, the first occurrence of the expression “feeding mechanism” should be replaced by\nfeeding apparatus.\nIn the Introduction, replace “the modern-type life history mode” by “one of the modern-type life\nhistory modes”. There are four modes of life in extant lampreys: blood feeders (parasites), flesh\nfeeders (predators), blood and flesh feeders (intermediates), and a presumed carrion feeder\n(scavenger) (Renaud and Cochran 2019). The full reference is: Renaud C.B. and P.A. Cochran. 2019.\nPost-metamorphic feeding in lampreys. pp. 247-285. In: Lampreys: Biology, Conservation, and\nControl. vol. 2, M.F. Docker (ed.), Fish and Fisheries Series 38, Springer Nature B.V.\nhttps://doi.org/10.1007/978-94-024-1684-8_3\nAt the end of the Introduction, change “renewed” to “revised” lamprey crown-group\nphylogeny.\nUnder Systematic Palaeontology, the oral disc size is not admissible as a diagnostic character for the\nnew genus because only one of the two new species has a measurable oral disc (see\nSupplementary Table 1). Notwithstanding, the oral disc is stated to be relatively small (at least in Y. occisor);\ncompared to what? The oral disc size should be quantified and compared. How do you know that the\nteeth are made of keratin? It is sufficient to say posterior disc teeth lacking (i.e., to include and\n“posterior” circumoral is implicit). I agree that the anterior and lateral circumoral teeth are spatulate\nin shape, but cannot understand what is meant by “and each bearing three convergent blades”.\nFigs 2d and f appear to be mislabeled. According to the species’ diagnoses, Fig. 2d would correspond\nto a drawing of Y. ingensdentes and Fig. 2f to a drawing Y. occisor. In the figure caption, however, both\nFigs 2d and f are stated to correspond to Y. occisor, respectively, as a photograph and a restoration.\nI disagree with the limits of the anterior and lateral fields delineated in Fig. 2g. According to Renaud\n(2011: 7) (reference 11 in the manuscript), the boundaries are set by the anteriormost edge of the\nsupraoral lamina and a line drawn through the apices of the two lateralmost cusps on the infraoral\nlamina. These limits, as defined by Renaud (2011), have repeatedly been used in the literature for\ncounts of lamprey teeth, including those of Geotria, which the authors state resemble those of\nYanliaomyzon.\nIn the diagnosis for Y. occisor, the tail region is stated to measure about one fourth of the total body\nlength, but according to Supplementary Table 1, this rather equals 38.7%! (i.e., a-C/TL = 270.7/700\n= 38.7% > 25.0%).\nIn the etymology for Y. ingensdentes, I would change rasping piston to gouging piston, because the\nlatter is the action of a flesh feeder, while the former is the action of a blood feeder.\nIn the diagnosis of Y. ingensdentes, the dorsal fin is said to be contiguous with the high and large\nanterior caudal fin, but I believe the interpretation of the fins is incorrect. Rather, the low first dorsal\nfin is contiguous with the high second dorsal fin and the latter is continuous with the caudal fin.\nIn the Description and comparison, it is stated that Y. occisor is > 700 mm, but Supplementary Table 1\nindicates exactly 700 mm, and therefore, the symbol greater than (i.e., >) should be deleted. This\nnew species is said to be only slightly smaller than P. marinus (1,200 mm) and E. tridentatus (850\nmm), but in this group should also be included G. australis (788 mm) and L. camtschaticum (790 mm)\n(see Renaud and Cochran 2019). Given these data, the qualifier “slightly” is incorrect and should be\nremoved. The relatively small oral discs of Y. occisor and Y. ingensdentes are stated to be respectively,\n4.8% and 6.7% of total length, but the former is correctly 4.7% and I can’t find the data to derive the\nlatter because Supplementary Table 1 does not have a value for the disc length of Y. ingensdentes. To\nstate that the oral disc length to total body length of the two Yanliaomyzon species approach the\ncondition of modern flesh-eating lampreys is not informative without providing the data. The data in\nPotter and Hilliard (1987) give a range for this ratio of 2.7–6.3% for flesh feeders (and specifically,\n2.7–3.2% in Geotria) and 5.8–11.8% for blood feeders. As mentioned above, to state that posterior\ndisc teeth are missing is sufficient because posterior circumoral teeth are by definition disc teeth. As\nalso mentioned above, the meaning of the longest of the three converging blades facing slightly\nsideways is unclear to me. The expression tricuspid cusps is nonsensical (a tooth or a lamina may be\ntricuspid, but not a cusp). It would make more sense to say The tips of these three cusps …\nThe prebranchial region is stated to be longer than the branchial apparatus in the two fossil lampreys;\nand this is certainly borne out in Y. ingensdentes (Supplementary Table 1: d–B1 = 42 versus B1–B7 =\n12), but neither of these morphometric characters is recorded in Y. occisor. The eyes are stated to be\nmedium-sized, but while a value of 4 is recorded in Supplementary Table 1 for Y. ingensdentes, none\nis given for Y. occisor.\nThe gular pouch is supposed to present in Fig. 2c and in Extended Data Fig. 2k, but this is certainly\nnot evident to me. It is present in Fig. 2e, but only indicated with the abbreviation g and is missing\nthe associated letter p, as defined in the anatomical abbreviations. According to references 11 and 22,\nthere are in fact 11 (not nine) living lamprey species with a gular pouch, and six (not five) of these are\nanadromous. However, you also need to add Geotria macrostoma; making 12 species with a gular\npouch, seven of which are anadromous. The reference for G. macrostoma is: Baker, C.F., C. Riva Rossi,\nP. Quiroga, E. White, P. Williams, J. Kitson, C.M. Bice, C.B. Renaud, I. Potter, F.J. Neira, and C. Baigún.\n2021. Morphometric and physical characteristics distinguishing adult Patagonian lamprey, Geotria\nmacrostoma from the pouched lamprey, Geotria australis. PLoS ONE 16(5): e0250601.\nhttps://doi.org/10.1371/journal.pone.0250601\nInstead of Before the cloaca, a long preanal skin fold …, I suggest A long precloacal skin fold …\nI suggest intestinal tract rather than intestinal duct.\nUnder Feeding mode, life history and swimming performance of Yanliaomyzon change stomach to\nintestinal or gut content.\nThe sentence This implies for the anadromy wherein the higher marine productivity … must be missing\nsome words because it makes no sense.\nAs suggested above, the preanal skin fold should be called precloacal skin fold. The long-based dorsal\nfin and ribbon-like precloacal skin fold considerably extend rather than extends.\nUnder The evolution of lampreys’ feeding structures and its implications, I do not understand what is\nmeant by hitch-hiking on other delivers. I suspect the word delivers is not what you mean.\nI would term the radical metamorphic phase, a stage rather than a phase.\nUnder The early biogeographical history of modern lampreys, shouldn’t Late Jurassic and Late\nCretaceous be periods instead of eras?\nI believe that the word promoted in relation to trans-equatorial dispersal is inappropriate and should\nmaybe be replaced by led to.\nI question the relevance of reference no. 48 in relation to major fish hosts of modern lampreys\nbecause in their revision of post-metamorphic feeding in lampreys, Renaud and Cochran (2019) do\nnot mention the genus Trisopterus as a host of lampreys.\nUnder Anatomical abbreviations, the adf, anterior dorsal fin should be called first dorsal fin and the\npdf, posterior dorsal fin should be called second dorsal fin. How can the posterior dorsal fin be\nsynonymous with the anterior part of the caudal fin? I note that in lines 237-240 of the\nSupplementary Information, the authors have justified their position in regards to dorsal and caudal\nfin definitions, but to me it remains unsatisfactory. While in Petromyzontidae the second dorsal fin\n(all genera minus Ichthyomyzon) or posterior lobe of the single dorsal fin (Ichthyomyzon) is\nconfluent with the caudal fin, Geotria australis is known to have a distinct second or posterior dorsal\nfin, separated by an interspace from the caudal fin.\nNote that in reference no. 28, the second author is Gill, not Gills.\nNote that in reference no. 34, the word Environments should have its first letter in lower case.\nNote that in reference no. 42, the word Pacific should have its first letter in upper case.\nFigs 3 and 4 contain much valuable information, but are too small for readers to be able to discern\nmany of the details and should be increased in size. In regards to Fig. 4, it is unclear to me to which\nfossil taxa the four red spots refer to. Additionally, the distinction between the light blue and dark\nblue shadows is undiscernible.\nThe title of Supplementary Table 1 is misleading and confusing. A meristic measurement is a\nmisnomer. The only meristic character it contains is the number of trunk myomeres and this has\nonly been determined in Mesomyzon mengae. I suggest deleting Meristic from the title and simply\nstarting with Measurements and removing the column number of trunk myomeres. I presume that all\nmeasurements are in mm, but this should be specified.\nFor completeness and balance of opinion, I strongly recommend the inclusion of two recent papers\ninto the Discussion; namely, Brownstein and Near (2023) and Mallatt (2022). The complete\nreferences are: Brownstein, C.D. and T.J. Near. 2023. Phylogenetics and the Cenozoic radiation of lampreys.\nCurrent Biology 33: 1-8; Mallatt, J. 2022. Vertebrate origins are informed by larval lampreys\n(ammocoetes): a response to Miyashita et al., 2021. Zoological Journal of the Linnean Society XX: 1-\n35.\nClaude B. Renaud", "reviewer_2": "This paper presents a radical revision of the evolutionary and ecological history of lampreys based on\nan exceptionally preserved fossil from the Jurassic. Prior fossils had suggested that the feeding\nmorphology of lamprey was highly constrained for the last 360 million years with the exception of the\npouched lamprey, and that key features like ammocete larvae evolved at some unknown time. The\npresent study uses new data from a Jurassic fossil to support an abrupt change in lamprey ecology\naround the Mesozoic origin of the crown, with flesh-eating as the ancestral state and greater diversity\nin feeding morphology than anticipated. The study is very well written and wide ranging and thorough\nin its methods, implications for the evolution of fishes, and in the number of factors considered as\ndrivers for the diversification of lamprey.\nHowever, the one, somewhat glaring, omission is a lack of consideration of conodonts, which recently\nhave been assigned to the cyclostome stem in other studies of fossil cyclostomes. Conodont were\nflesheaters with feeding apparati that sometimes resemble the novel plates of the new lamprey.\nThey also finally went extinct at the end-Triassic. It seems possible that the extinction of sometimes\nlarge-bodied (>1m), pelagic, shearing-biting conodonts may have opened space for the previously\nnon-biting lamprey (although conodonts were never freshwater). Conodonts are often ignored in\nevolutionary studies of fishes, but in this case there could be a real influence on the timing of lamprey\ndiversification and the change in their ecology proposed here. I would like to see some discussion of\nthis possibility.\nIn any case, this discovery and resultant investigation raises a number of interesting possibilities for\nand questions about Mesozoic fish evolution, which will lead to further reconsideration of the record\nof lamprey and their ecosystems. I look forward to seeing it published in Nature Communications after\nminor revisions to the discussion about conodonts.\nLauren Sallan", "reviewer_3": "GENERAL COMMENTS:\nThis manuscript describes two very well-preserved large lampreys, each identified as a new species,\nfrom the Middle-Late Jurassic Yanliao Biota of North China, dating to approximately 163-158 million\nyears ago. Their very large body size (especially Yanliaomyzon occisor, which exceeds 700 mm in\nlength) is far greater than any fossil lamprey yet described and is on par with some of the largest\nmodern anadromous lampreys. This find is very exciting, particularly given the recent study by\nMiyashita et al. (2021) showing that the ammocoete stage in lampreys is a derived characteristic,\nand the presumption by previous authors (e.g., Evans et al. 2018; Docker and Potter 2019, attached) that,\nfollowing the evolution of a radical metamorphosis, the ammocoete larval stage and the post-\nmetamorphic juvenile stage subsequently became well-adapted to their respective environments and\nthe growth potential of each period was maximized, enabling the large body size that now\ncharacterizes modern parasitic lampreys. The fact that these hypotheses have has now been\ndemonstrated and dated in the fossil record is very exciting. As other authors have pointed out,\nlampreys have maintained a conserved morphology over hundreds of millions of years, but the\nevolution of the modern lamprey life history has been more recent and less well understood.\nThe conclusion that the phylogenetic distribution of the feeding modes suggests that the flesh-eating\nhabit should be ancestral for modern lampreys is likewise novel and interesting. The finding of\npresumed intestinal contents of several tooth-bearing jaw bones and possibly skull bones of some\nunidentified bony fishes was particularly interesting, countering previous suggestions that, among\nmodern parasitic lampreys, blood feeding is ancestral (Potter and Hilliard 1987). Potter and Hilliard\n(1987) argued that blood feeding would be less detrimental to the hosts (and thus, ultimately, to the\nlampreys which depend on them) in less productive waters. Blood is a renewable resource if the rate\nof feeding is not excessive relative to the size of the host, and they concluded that flesh feeding came\nlater, with access to smaller but more plentiful coastal fishes (e.g., herrings and other clupeids) or\nwhere lampreys travel farther offshore to feed in very productive waters. I think readers would be able\nto appreciate the significance of your new findings more with a fuller explanation of previous views.\nThe resemblance to the Southern Hemisphere pouched lamprey was also interesting although, given\nthat dentition in lampreys may evolve convergently in different taxa (e.g., the dentition of Lampetra\nayresii from western North American is virtually indistinguishable from the dentition of Lampetra\nfluviatilis from western Europe, even though they do not form a monophyletic group), I found the\nconclusion that modern lampreys originated in the Southern Hemisphere somewhat less definitive.\nNevertheless, this is another valuable contribution to our understanding of lamprey evolution.\nHowever, although all these findings are very exciting, I found that the discussion of the manuscript\ndid not highlight them sufficiently. I found the discussion was less concise and less focused than it\ncould have been, which, in my opinion, diluted and distracted from the novel and exciting results.\nSome suggestions for improvement are below.\nOverall, these fossils represent a VERY exciting discovery. With revision, I think the manuscript is\nsuitable for publication in Nature Communications. With a discussion that more concisely highlights\nthe importance of these findings and better places them in the context of what we already know or\nhave surmised about the evolution of lamprey life history type, I think it would be of considerable\ninterest to both a broad audience and lamprey biologists specifically.\nSPECIFIC COMMENTS:\nLine 4 – Virtually all authorities now recognize lampreys and hagfishes as a monophyletic group,\nmaking lampreys and hagfishes equally “the oldest living jawless vertebrates”\nLine 61, 62 – What classification are you following? Please indicate. Recent classifications with which I\nam familiar (e.g., Nelson et al. 2016, Fishes of the World, 5th edition) place hagfishes and lampreys\ninto separate classes (Myxini and Petromyzontida, respectively) and I have not seen Hyperoartii used\nas a synonym for order Petromyzontiformes.\nLine 110 – Do modern flesh-eating lampreys have smaller oral discs relative to blood-sucking\nlampreys (i.e., are oral discs 4.8% and 6.7% of total body length inconsistent with blood-feeding)?\nPlease be explicit.\nLine 138-139 – Are you including Geotria macrostoma in your enumeration? Presumably yes since you\ncite Riva-Rossi et al. (2020; citation 27) in this manuscript, but both references that you cite with\nrespect to the gular pouch (Monette and Renaud 2005; Renaud 2011) were published before the\nrevalidation of Geotria macrostoma; if you’ve taken the information directly from Monette and Renaud\n(2005) and Renaud (2011), it won’t be up to date.\nLine 188-192 – This paragraph seems insufficient for a broader audience unfamiliar with lamprey\nbiology while also coming across as somewhat superficial for audiences with a familiarity of\ndifferent lamprey/fish life history types. This is particularly the case for the sentences “In body\nlength, Yanliaomyzon occisor falls within modern anadromous lampreys but notably departs from the\nsmaller freshwater residents and non-feeding forms5. This implies for the anadromy wherein the higher\nmarine productivity could have provided more feeding opportunities and hence greater growth\npotential.” It has already been stated in this manuscript (Line 101) that these two species are among\nthe largest of modern anadromous lampreys, and the differences in growth potential between\nanadromous and freshwater fishes (not just lampreys) is generally well known, so the suggestion\nhere that higher marine productivity could have provided more feeding opportunities and hence\ngreater growth potential isn’t original. It would seem more appropriate to state that the large body size\nhere is consistent with known larger body size in anadromous fishes compared to their freshwater\ncounterparts or that, for the first time, your findings provide evidence of this predicted transition to\nanadromy and have been able to date it. Placing your new findings in the context of what is already\nknown or what has already been predicted about the evolution of lamprey life history type would make\nthis more interesting. Also, the term “non-feeding forms” might be misleading to those unfamiliar with\nnon-parasitic lampreys since they are only non-feeding after the onset of metamorphosis.\nLine 192-195 – It is not clear here if you are suggesting that Yanliaomyzon evolved a triphasic life\ncycle or rather that it inherited it from the common ancestor that possessed a triphasic life cycle. I\nthink the latter, but the wording could suggest the former.\nLine 221 – The meaning of “which should be historical products of the enhancement of the feeding\nstructures” is not clear to me\nLine 234-254 – As with Line 188-192, this section seems rather simplistic, neither clearly explaining\nthe “big picture” for a broad audience nor concisely and clearly describing what is novel here for a\nmore expert audience.\nEDITORIAL COMMENTS:\nThe language used sometimes appears too sensational or not quite appropriate, for example:\nLine 5, 29 – “bizarre” feeding behavior\nLine 6, 29 – “gouging out” tissues from their “victims”\nLine 38 – “renovated” the life history strategy\nLine 127 – “tricuspid cusps”\nLine 179-180 – a large, “monstrous” flesh eater that can even “destroy” the skull of teleost fish\nSuggested corrections:\nLine 163 – petromyzontiform history (instead of petromyzontiform’s history), assuming that Mesozoic\nlampreys are placed in the same order as extant lampreys. Is that the case?\nTypographical error noted:\nLine 387 – Gills should be Gill"}, "author_response": "RESPONSES TO REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis is an outstanding contribution to our knowledge of lamprey phylogeny and biogeography\nthrough the integration of all known fossil and virtually all extant lampreys (Geotria macrostoma\nexcluded) (Re: Yes, despite of the differences in the details of the teeth and some ecological\nfeatures, there is a consensus that Geotria macrostoma and Geotria australis are most closely\nrelated species (relevant refs cited in our study), herein we choose the latter as the\nrepresentative of this genus as its morphological data is currently more complete than the\nother) that feed post-metamorphosis. The discovery of two new fossil lampreys has enabled this\nbreakthrough.\nI have a number of comments and suggested changes to improve the manuscript.\nThe authors refer to the Jurassic era, but shouldn’t it be Jurassic Period? (Re: Rephrased already)\nIn the Abstract, the three phases of the life cycle should be specified for clarification. (Re: We have\nrephrased this part and specified the three phases of living lampreys’ life cycle)\nThe word “hosts” at the end of the Abstract and Introduction should be replaced by prey because,\ngiven their oral and lingual dentitions, the two newly-described fossil lamprey species were more\nlikely predators rather than parasites. (Re: Thanks for your suggestion. We have revised the\nwords for lampreys’ hosts (for blood-feeders) or preys (for flesh-feeders) in the text.\nHowever, we referred ‘hosts’ to the lineage represented by Mesomyzon which was estimated\nto stem from the Early Jurassic and is likely a blood-feeder (referring to ref. 9, our restudy of\nMesomyzon judged by its body size and the features of its suctorial oral disc). In other words,\nwe think the earliest lampreys with modern-type life cycle should be blood-feeding when other\nfishes evolved thinned integument (from enameled scales in primitive forms to bony cycloid\nscales of advanced teleosts))\nIn the Abstract, the word “the” should precede Pangean supercontinent.(Re: Corrected.)\nIn the Introduction, it is unclear to me what is meant by “simply assembled analogue”. This needs\nto be clarified.( Re: We referred to their simply assembled teeth, and this part has been\nrephrased)\nIn the Introduction, the first occurrence of the expression “feeding mechanism” should be replaced\nby feeding apparatus. (Re: Yes, we have replaced “feeding mechanism” with “feeding\napparatus” in the text)\nIn the Introduction, replace “the modern-type life history mode” by “one of the modern-type life\nhistory modes”. There are four modes of life in extant lampreys: blood feeders (parasites), flesh\nfeeders (predators), blood and flesh feeders (intermediates), and a presumed carrion feeder\n(scavenger) (Renaud and Cochran 2019). The full reference is: Renaud C.B. and P.A. Cochran. 2019.\nPost-metamorphic feeding in lampreys. pp. 247-285. In: Lampreys: Biology, Conservation, and\nControl. vol. 2, M.F. Docker (ed.), Fish and Fisheries Series 38, Springer Nature\nB.V. https://doi.org/10.1007/978-94-024-1684-8_3 (Re: Thanks for providing the reference\nwhich has been cited in the revised text. In fact, here we referred to the life cycle (from\nammocoete to adult) mode, not living or feeding mode)\nAt the end of the Introduction, change “renewed” to “revised” lamprey crown-group\nphylogeny.(Re: Corrected)\nUnder Systematic Palaeontology, the oral disc size is not admissible as a diagnostic character for\nthe new genus because only one of the two new species has a measurable oral disc (see\nSupplementary Table 1) (Re: Indeed, the oral disc of the holotype of Y. ingensdentes was\npartially distorted and cannot be accurately measured, therefore the measurements in S Table\n1 have been revised accordingly). Notwithstanding, the oral disc is stated to be relatively small (at\nleast in Y. occisor); compared to what? (Re: The ratio of the relative size of the oral disc of Y.\noccisor falls within the ratio of living flesh-feeding lampreys which is larger than that of the\ntypical blood-feeding species, please also check the replies below and the revised text for this\nissue) The oral disc size should be quantified and compared. How do you know that the teeth are\nmade of keratin? (Re: Technically, we failed to determine the nature of the teeth of our fossils,\nas we conducted the element analyses via electronic scanning, we only detected Si element in\nthe position of the teeth, which suggests that the teeth might have been replaced by the mineral\nelements from the rock matrix (This implies for some special preservation settings. Notably,\nsome flying dinosaurs with membranous wings were discovered from the same layers). For\nnow, we can just take them as the analogues of keratinous teeth in living lampreys.) It is\nsufficient to say posterior disc teeth lacking (i.e., to include and “posterior” circumoral is implicit).\nI agree that the anterior and lateral circumoral teeth are spatulate in shape, but cannot understand\nwhat is meant by “and each bearing three convergent blades” (Re: Admittedly, the description\nwas not clear enough. Each circumoral tooth looks like a trihedron that bears three edges of\ndifferent lengths. We have replaced the reconstructions of the oral discs and dentition of our\nfossil lampreys (Fig. 2d, f ) with high-quality computer-generated images that will illustrate\nthe anatomy much better).\nFigs 2d and f appear to be mislabeled (Re: The mislabelings have been corrected; as for the\ndescription of the circumoral teeth, we meant that each tooth looks like a trihedron with the\ntip pointing to the direction of the oral aperture; we have revised Fig. 2d, f to illustrate more\nclearly the morphology of these teeth）\nAccording to the species’ diagnoses, Fig. 2d would correspond to a drawing of Y. ingensdentes and\nFig. 2f to a drawing Y. occisor (Re: Yes, these figs were mislabeled and the figure legend has\nbeen fixed). In the figure caption, however, both Figs 2d and f are stated to correspond to Y. occisor,\nrespectively, as a photograph and a restoration (Re: Corrected).\nI disagree with the limits of the anterior and lateral fields delineated in Fig. 2g. According to Renaud\n(2011: 7) (reference 11 in the manuscript), the boundaries are set by the anteriormost edge of the\nsupraoral lamina and a line drawn through the apices of the two lateralmost cusps on the infraoral\nlamina. These limits, as defined by Renaud (2011), have repeatedly been used in the literature for\ncounts of lamprey teeth, including those of Geotria, which the authors state resemble those of\nYanliaomyzon. (Re: Yes, we have corrected the limits of the anterior and lateral fields of oral\ndisc teeth in Fig. 2g according to your suggestions and relevant literatures)\nIn the diagnosis for Y. occisor, the tail region is stated to measure about one fourth of the total body\nlength, but according to Supplementary Table 1, this rather equals 38.7%! (i.e., a-C/TL = 270.7/700\n= 38.7% > 25.0%).( Re: Yes, indeed! Thank you for pointing out these problems. We measured\nagain the holotype and corrected some measurements. Now we calculated that the tail length\noccupies ca. 28% of total body length, which is still much shorter than that in Y. ingensdentes\n(slightly more than 40 %)).\nIn the etymology for Y. ingensdentes, I would change rasping piston to gouging piston, because the\nlatter is the action of a flesh feeder, while the former is the action of a blood feeder. (Re: Yes, we\nhave replaced ‘rasping’ piston with ‘gouging’ piston for this flesh-eating lamprey) In the\ndiagnosis of Y. ingensdentes, the dorsal fin is said to be contiguous with the high and large anterior\ncaudal fin, but I believe the interpretation of the fins is incorrect. Rather, the low first dorsal\nfin is contiguous with the high second dorsal fin and the latter is continuous with the caudal fin. (Re: Yes,\nwe get your points. Actually, different from the conventional terminology for lampreys’ fins,\nwe (Wu et al., 2021, [p.1298 in ref. 8 in current study]; Janvier, 2008 [p.1051 in ref. 2 in current\nstudy]) ever tentatively homologized the ‘posterior dorsal fin’ with the anterior part of dorsal\nlobe of the caudal fin that is mostly located behind the level of the cloaca (In fishes, this opening\nmarks the boundary of the abdominal and caudal regions). Additionally, regarding the\ndistribution of the cartilaginous radials of the fins, we also noted that those of the ‘posterior\ndorsal fin’ and the caudal fin are always continuous in the vast majority of living lampreys\n(with the exception of Geotria) (Renaud, 2011), probably reflecting a closer relation between\nthese two parts than that between the anterior (first) and the conventionally-termed posterior\n(second) dorsal fins. Especially when we have to compare lampreys with other early\nvertebrates (including those fossil jawless vertebrates in Devonian, e.g., the ‘naked anaspids’\n(see Janvier, 2008, ref. 2 in current study)) from an evolutionary perspective, we will\nencounter the problem of the homology of the dorsal median fins of early vertebrates. Our\nhypothesis might be an alternative solution.)\nIn the Description and comparison, it is stated that Y. occisor is > 700 mm, but Supplementary Table\n1 indicates exactly 700 mm, and therefore, the symbol greater than (i.e., >) should be deleted. This\nnew species is said to be only slightly smaller than P. marinus (1,200 mm) and E. tridentatus (850\nmm), but in this group should also be included G. australis (788 mm) and L. camtschaticum (790\nmm) (see Renaud and Cochran 2019). Given these data, the qualifier “slightly” is incorrect and\nshould be removed. (Re: Yes, we have measured again the fossils and included all these large\nliving species in the text and deleted the qualifier“slightly”. The ref. you referred to has been\ncited in the revised text.)\nThe relatively small oral discs of Y. occisor and Y. ingensdentes are stated to be respectively, 4.8%\nand 6.7% of total length, but the former is correctly 4.7% and I can’t find the data to derive the latter\nbecause Supplementary Table 1 does not have a value for the disc length of Y. ingensdentes (Re:\nThank you for pointing out these problems. We rechecked the fossils and fixed the\nmeasurements of Supplementary Table I. The oral disc of the holotype of Y. ingensdentes was\npartially distorted and cannot be accurately measured, therefore the measurements in S Table\n1 was revised accordingly. We also rechecked the data of Y. occisor and revised some\nmeasurements of this species). To state that the oral disc length to total body length of the two\nYanliaomyzon species approach the condition of modern flesh-eating lampreys is not informative\nwithout providing the data. The data in Potter and Hilliard (1987) give a range for this ratio of 2.7–\n6.3% for flesh feeders (and specifically, 2.7–3.2% in Geotria) and 5.8–11.8% for blood feeders. (Re:\nWe measured the oral disc size of Yanliaomyzon occisor again and corrected the numbers in\nthe text and S Table 1)\nAs mentioned above, to state that posterior disc teeth are missing is sufficient because posterior\ncircumoral teeth are by definition disc teeth. As also mentioned above, the meaning of the longest\nof the three converging blades facing slightly sideways is unclear to me (Re: We have revised Fig.\n2d, f and the description to show more clearly the structure of the circumoral teeth).\nThe expression tricuspid cusps is nonsensical (a tooth or a lamina may be tricuspid, but not a cusp).\nIt would make more sense to say The tips of these three cusps (Re: Thanks, the text here has been\ncorrected)\nThe prebranchial region is stated to be longer than the branchial apparatus in the two fossil lampreys;\nand this is certainly borne out in Y. ingensdentes (Supplementary Table 1: d–B1 = 42 versus B1–\nB7 = 12), but neither of these morphometric characters is recorded in Y. occisor. (Re: Indeed, we\nmissed some values for Y. occisor in the table. Actually, for these values we measured the\nparatype (IVPP V 18956B) of Y. occisor (Fig. 1c, d) where the impression of the head and\nbranchial region was preserved but not in the holotype. The numbers now have been added\nin Supplementary Table 1)\nThe eyes are stated to be medium-sized, but while a value of 4 is recorded in Supplementary Table\n1 for Y. ingensdentes, none is given for Y. occisor. (Re: Indeed, for this value we measured the\nparatype of Y. occisor (Fig. 1c, d) and added the value in Supplementary Table 1)\nThe gular pouch is supposed to present in Fig. 2c and in Extended Data Fig. 2k, but this is certainly\nnot evident to me (Re: Yes, this structure is not completely preserved. However, according to\nthe part (Fig. 2a) and counterpart (Fig. 2c, Extended Data Fig. 2k, with the gular pouch better\npreserved than in Fig. 2a)) of the specimen, we saw a depression with a curving lateral edge\n(Fig. 2a, b) that matches the pouch-like protrusion (comparable to the preservation state of\nthe gular pouch in Y. occisor (see Fig. 1b)). Despite lacking the ventral part, the remaining\npart of this protrusion shows a pouch-like profile (compare to the preserved gular pouch of Y.\noccisor [Fig. 1b]) and bears some wrinkles produced by the tissues of gular pouch\n(pointed by the white arrow in Fig. 2c). It is present in Fig. 2e, but only indicated with the\nabbreviation g and is missing the associated letter p, as defined in the anatomical abbreviations (Re:\nWe have rechecked the material of Fig. 2e and confirmed the large structure with a round\nventral edge and three tips which was delineated as the ‘transverse lingual lamina’ [‘tl’]; we\nhave also checked and added the labeling of the gular pouch (gp) in the revised Fig. 2c).\nAccording to references 11 and 22, there are in fact 11 (not nine) living lamprey species with a gular\npouch, and six (not five) of these are anadromous (Re: Yes, indeed, we have checked and\nrephrased the text here). However, you also need to add Geotria macrostoma; making 12 species with a gular\npouch, seven of which are anadromous. The reference for G. macrostoma is: Baker,\nC.F., C. Riva Rossi, P. Quiroga, E. White, P. Williams, J. Kitson, C.M. Bice, C.B. Renaud, I. Potter,\nF.J. Neira, and C. Baigún. 2021. Morphometric and physical characteristics distinguishing adult\nPatagonian lamprey, Geotria macrostoma from the pouched lamprey, Geotria australis. PLoS ONE\n16(5): e0250601. https://doi.org/10.1371/journal.pone.0250601 (Re: Yes, we have included this\nreference in the text and revised the text therein)\nInstead of Before the cloaca, a long preanal skin fold …, I suggest A long precloacal skin fold …\n(Revised)\nI suggest intestinal tract rather than intestinal duct. (Revised)\nUnder Feeding mode, life history and swimming performance of Yanliaomyzon change stomach to\nintestinal or gut content.(Revised)\nThe sentence This implies for the anadromy wherein the higher marine productivity … must be\nmissing some words because it makes no sense.(Re: The sentence has been rephrased.)\nAs suggested above, the preanal skin fold should be called precloacal skin fold. The long-based\ndorsal fin and ribbon-like precloacal skin fold considerably extend rather than extends.( Re: We\nhave revised this part of description according to your suggestion)\nUnder The evolution of lampreys’ feeding structures and its implications, I do not understand what\nis meant by hitch-hiking on other delivers. I suspect the word delivers is not what you\nmean.(Revised)\nI would term the radical metamorphic phase, a stage rather than a phase.(Corrected)\nUnder The early biogeographical history of modern lampreys, shouldn’t Late Jurassic and Late\nCretaceous be periods instead of eras? (Revised)\nI believe that the word promoted in relation to trans-equatorial dispersal is inappropriate and should\nmaybe be replaced by led to.(Revised)\nI question the relevance of reference no. 48 in relation to major fish hosts of modern lampreys\nbecause in their revision of post-metamorphic feeding in lampreys, Renaud and Cochran (2019) do\nnot mention the genus Trisopterus as a host of lampreys.( Re: Thank you for reminding us of the\nreference and the book (volume 2 of ‘Lampreys: Biology, Conservation and Control’) that\nincluded this paper. We have gone through carefully these references and agree with you that\nwe should exclude Trisopterus from list of the host victims of lampreys.)\nUnder Anatomical abbreviations, the adf, anterior dorsal fin should be called first dorsal fin and the\npdf, posterior dorsal fin should be called second dorsal fin. How can the posterior dorsal fin be\nsynonymous with the anterior part of the caudal fin? I note that in lines 237-240 of the\nSupplementary Information, the authors have justified their position in regards to dorsal and caudal\nfin definitions, but to me it remains unsatisfactory. While in Petromyzontidae the second dorsal fin\n(all genera minus Ichthyomyzon) or posterior lobe of the single dorsal fin (Ichthyomyzon) is\nconfluent with the caudal fin, Geotria australis is known to have a distinct second or posterior dorsal\nfin, separated by an interspace from the caudal fin (Re: Thanks. The ‘confluence’ between the\n‘second dorsal fin’ and the caudal fin in stem lampreys, as well as in the majority of living\nlampreys suggests that this state should be ancestral of crown lamprey lineages. Geotria’s\ndisjunction of these two parts is a derived feature). (For this issue, we responded to your\ncomments above. We reinterpreted the conventionally-termed ‘posterior dorsal fin’ as the\nanterior part of the caudal fin according to its relative position to the cloaca and a broader\ncomparison to early jawless vertebrates from an evolutionary perspective).\nNote that in reference no. 28, the second author is Gill, not Gills.(Corrected)\nNote that in reference no. 34, the word Environments should have its first letter in lower case.\n(Corrected)\nNote that in reference no. 42, the word Pacific should have its first letter in upper case. (Corrected)\nFigs 3 and 4 contain much valuable information, but are too small for readers to be able to discern\nmany of the details and should be increased in size. In regards to Fig. 4, it is unclear to me to which\nfossil taxa the four red spots refer to. Additionally, the distinction between the light blue and dark\nblue shadows is undiscernible. (Re: Thank you for your suggestions. We have reorganized Figs\n3 and 4, increasing the size and the color contrast between different units. Also, we have added\npanels in Fig. 4 to illustrate the results of the palaeobiogeographical analysis and show the\npossible ancestral geographical distributions for some relevant nodes, e.g., that of the crown-\ngroup lampreys.)\nThe title of Supplementary Table 1 is misleading and confusing. A meristic measurement is a\nmisnomer. The only meristic character it contains is the number of trunk myomeres and this has\nonly been determined in Mesomyzon mengae. I suggest deleting Meristic from the title and simply\nstarting with Measurements and removing the column number of trunk myomeres. I presume that\nall measurements are in mm, but this should be specified. (Re: Yes, we have revised the title of\nthe table, added ‘all measurements are in mm’ and deleted the column of the number of trunk\nmyomeres)\nFor completeness and balance of opinion, I strongly recommend the inclusion of two recent papers\ninto the Discussion; namely, Brownstein and Near (2023) and Mallatt (2022). The complete\nreferences are: Brownstein, C.D. and T.J. Near. 2023. Phylogenetics and the Cenozoic radiation of\nlampreys. Current Biology 33: 1-8; Mallatt, J. 2022. Vertebrate origins are informed by larval\nlampreys (ammocoetes): a response to Miyashita et al., 2021. Zoological Journal of the Linnean\nSociety XX: 1-35.( Re: Thank you for providing the information of these important literatures.\nWe have included these refs in the revised MS and rewritten the relevant part of the text.\nWe noticed that although Brownstein, C.D. and T.J. Near. (2023）highlighted the Cenozoic\nradiation of the lampreys in the Northern Hemisphere, their topology of the phylogeny and\nthe timing of the node of the crown-group lampreys are radically different from those in our\nstudy.)\nClaude B. Renaud\nReviewer #2 (Remarks to the Author):\nThis paper presents a radical revision of the evolutionary and ecological history of lampreys based\non an exceptionally preserved fossil from the Jurassic. Prior fossils had suggested that the feeding\nmorphology of lamprey was highly constrained for the last 360 million years with the exception of\nthe pouched lamprey, and that key features like ammocete larvae evolved at some unknown time.\nThe present study uses new data from a Jurassic fossil to support an abrupt change in lamprey\necology around the Mesozoic origin of the crown, with flesh-eating as the ancestral state and greater\ndiversity in feeding morphology than anticipated. The study is very well written and wide ranging\nand thorough in its methods, implications for the evolution of fishes, and in the number of factors\nconsidered as drivers for the diversification of lamprey.\nHowever, the one, somewhat glaring, omission is a lack of consideration of conodonts, which\nrecently have been assigned to the cyclostome stem in other studies of fossil cyclostomes. Conodont\nwere flesheaters with feeding apparati that sometimes resemble the novel plates of the new lamprey.\nThey also finally went extinct at the end-Triassic. It seems possible that the extinction of sometimes\nlarge-bodied (>1m), pelagic, shearing-biting conodonts may have opened space for the previously\nnon-biting lamprey (although conodonts were never freshwater). Conodonts are often ignored in\nevolutionary studies of fishes, but in this case there could be a real influence on the timing of\nlamprey diversification and the change in their ecology proposed here. I would like to see some\ndiscussion of this possibility. (Re: Thank you very much for providing this enlightening point.\nYou are definitely right, so we have to fix it, as the conodonts and lampreys ever cooccurred\nfor such a long geological history, are so closely related phylogenetically and are very similar\nin the overall morphology (body plan) and the functional anatomy of the oral apparatus\n(lingual lamina system), conodonts were probably the ecological competitors when they\ncoexisted with lampreys and their extinction in the latest Triassic must have arguably\n‘released’ new ecological niches for lampreys, which might have inspired the evolution of\nlampreys during the following Jurassic period, a crucial period of lampreys’ history as\nhighlighted in our study. Following your suggestions, we have added some comments and cited\ntwo relevant references in the text. The new refs are: (1) Ginot, S. and Goudemand N.\nConodont size, trophic level, and the evolution of platform elements. Paleobiology, 1-11 (2019).\ndoi: 10.1017/pab.2019.19\n(2) Iannicelli, M. Solving the mystery of endless life between conodonts and lampreys, plus a\nreason for final extinction of the conodonts. J Oceanogr Mar Res S1: 001(2017). doi:\n10.4172/2572-3103.S1-001)\nIn any case, this discovery and resultant investigation raises a number of interesting possibilities for\nand questions about Mesozoic fish evolution, which will lead to further reconsideration of the record\nof lamprey and their ecosystems. I look forward to seeing it published in Nature Communications\nafter minor revisions to the discussion about conodonts.\nLauren Sallan\nReviewer #3 (Remarks to the Author):\nGENERAL COMMENTS:\nThis manuscript describes two very well-preserved large lampreys, each identified as a new species,\nfrom the Middle-Late Jurassic Yanliao Biota of North China, dating to approximately 163-158\nmillion years ago. Their very large body size (especially Yanliaomyzon occisor, which exceeds 700\nmm in length) is far greater than any fossil lamprey yet described and is on par with some of the\nlargest modern anadromous lampreys. This find is very exciting, particularly given the recent study\nby Miyashita et al. (2021) showing that the ammocoete stage in lampreys is a derived characteristic,\nand the presumption by previous authors (e.g., Evans et al. 2018; Docker and Potter 2019, attached)\nthat, following the evolution of a radical metamorphosis, the ammocoete larval stage and the post-\nmetamorphic juvenile stage subsequently became well-adapted to their respective environments and\nthe growth potential of each period was maximized, enabling the large body size that now\ncharacterizes modern parasitic lampreys. The fact that these hypotheses have has now been\ndemonstrated and dated in the fossil record is very exciting. As other authors have pointed out,\nlampreys have maintained a conserved morphology over hundreds of millions of years, but the\nevolution of the modern lamprey life history has been more recent and less well understood. (Re:\nWe have revised the text and figures by improving the quality of the reconstructions of our\nfossil lampreys’ feeding apparatus (Fig. 2d, f), adding the results of ancestral state and\nbiogeographical reconstructions and mapping them on the renewed Figs. 3 & 4, please check\nthe revised MS, which should make our points clearer.)\nThe conclusion that the phylogenetic distribution of the feeding modes suggests that the flesh-eating\nhabit should be ancestral for modern lampreys is likewise novel and interesting. The finding of\npresumed intestinal contents of several tooth-bearing jaw bones and possibly skull bones of some\nunidentified bony fishes was particularly interesting, countering previous suggestions that, among\nmodern parasitic lampreys, blood feeding is ancestral (Potter and Hilliard 1987). Potter and Hilliard\n(1987) argued that blood feeding would be less detrimental to the hosts (and thus, ultimately, to the\nlampreys which depend on them) in less productive waters. Blood is a renewable resource if the rate\nof feeding is not excessive relative to the size of the host, and they concluded that flesh feeding\ncame later, with access to smaller but more plentiful coastal fishes (e.g., herrings and other clupeids)\nor where lampreys travel farther offshore to feed in very productive waters. I think readers would be able\nto appreciate the significance of your new findings more with a fuller explanation of previous\nviews. (Re: Thank you for your suggestions. We have reorganized and rephrased the text by\nadding the results of our ancestral state reconstructions of the feeding apparatus and mapping\nthem on a renewed Fig. 3, please check the revised MS)\nThe resemblance to the Southern Hemisphere pouched lamprey was also interesting although, given\nthat dentition in lampreys may evolve convergently in different taxa (e.g., the dentition of Lampetra\nayresii from western North American is virtually indistinguishable from the dentition of Lampetra\nfluviatilis from western Europe, even though they do not form a monophyletic group), I found the\nconclusion that modern lampreys originated in the Southern Hemisphere somewhat less definitive.\nNevertheless, this is another valuable contribution to our understanding of lamprey evolution. (Re:\nWe have reorganized and rephrased the text by adding the results of our biogeographical\nanalysis and mapping them on the lamprey timetree in the revised Fig. 4, please check the\nrevised MS)\nHowever, although all these findings are very exciting, I found that the discussion of the manuscript\ndid not highlight them sufficiently. I found the discussion was less concise and less focused than it\ncould have been, which, in my opinion, diluted and distracted from the novel and exciting results.\nSome suggestions for improvement are below. (Re: We have reorganized and rephrased the\nsection of discussion accordingly, please see the revised MS)\nOverall, these fossils represent a VERY exciting discovery. With revision, I think the manuscript is\nsuitable for publication in Nature Communications. With a discussion that more concisely highlights\nthe importance of these findings and better places them in the context of what we already know or\nhave surmised about the evolution of lamprey life history type, I think it would be of considerable\ninterest to both a broad audience and lamprey biologists specifically. (Re: We have rephrased the\ntext accordingly, please see the revised MS)\nSPECIFIC COMMENTS:\nLine 4 – Virtually all authorities now recognize lampreys and hagfishes as a monophyletic group,\nmaking lampreys and hagfishes equally “the oldest living jawless vertebrates”(Re: Revised. In\nthe former version, we referred to the known fossil records of these two jawless groups, in\nwhich lampreys have older fossils than hagfishes)\nLine 61, 62 – What classification are you following? Please indicate. Recent classifications with\nwhich I am familiar (e.g., Nelson et al. 2016, Fishes of the World, 5th edition) place hagfishes and\nlampreys into separate classes (Myxini and Petromyzontida, respectively) and I have not seen\nHyperoartii used as a synonym for order Petromyzontiformes. (Corrected.)\nLine 110 – Do modern flesh-eating lampreys have smaller oral discs relative to blood-sucking\nlampreys (i.e., are oral discs 4.8% and 6.7% of total body length inconsistent with blood-feeding)?\nPlease be explicit.( Re: The text here has been revised according to your suggestion. According\nto the study of Potter and Hilliard, 1987 (ref.10 of current study), this ratio is 2.7–6.3% for\nflesh feeders, and 5.8–11.8% for blood feeders, reflecting feeding mode of the blood feeders\nthat require the attachment on the host victims for longer time than the flesh feeders)\nLine 138-139 – Are you including Geotria macrostoma in your enumeration? Presumably yes\nsince you cite Riva-Rossi et al. (2020; citation 27) in this manuscript, but both references that you\ncite with respect to the gular pouch (Monette and Renaud 2005; Renaud 2011) were published\nbefore the revalidation of Geotria macrostoma; if you’ve taken the information directly from\nMonette and Renaud (2005) and Renaud (2011), it won’t be up to date.( Re: Thank you for\nreminding us of this. We have fixed it and also added a reference--Baker, C.F., C. Riva Rossi,\nP. Quiroga, E. White, P. Williams, J. Kitson, C.M. Bice, C.B. Renaud, I. Potter, F.J. Neira,\nand C. Baigún. 2021. Morphometric and physical characteristics distinguishing adult\nPatagonian lamprey, Geotria macrostoma from the pouched lamprey, Geotria australis. PLoS\nONE 16(5): e0250601)\nLine 188-192 – This paragraph seems insufficient for a broader audience unfamiliar with lamprey\nbiology while also coming across as somewhat superficial for audiences with a familiarity of\ndifferent lamprey/fish life history types. This is particularly the case for the sentences “In body\nlength, Yanliaomyzon occisor falls within modern anadromous lampreys but notably departs from\nthe smaller freshwater residents and non-feeding forms5. This implies for the anadromy wherein\nthe higher marine productivity could have provided more feeding opportunities and hence greater\ngrowth potential.” It has already been stated in this manuscript (Line 101) that these two species are\namong the largest of modern anadromous lampreys, and the differences in growth potential between\nanadromous and freshwater fishes (not just lampreys) is generally well known, so the suggestion\nhere that higher marine productivity could have provided more feeding opportunities and hence\ngreater growth potential isn’t original (Re: Yes, we agree with you. There are sufficient evidences\nthat those species and the populations that feed in marine ecosystem. We have added a\nreference-- Renaud CB and Cochran PA. Post-metamorphic Feeding in Lampreys. In Docker,\nM. F.ed. Lampreys: Biology, Conservation and Control, p.247-286 (Springer,2019)).\nIt would seem more appropriate to state that the large body size here is consistent with known larger\nbody size in anadromous fishes compared to their freshwater counterparts or that, for the first time,\nyour findings provide evidence of this predicted transition to anadromy and have been able to date\nit.( Re: Yes, we have rephrased this part) Placing your new findings in the context of what is\nalready known or what has already been predicted about the evolution of lamprey life history type\nwould make this more interesting (Re: Indeed, we have rewritten this part and noted the he\nconventional wisdom that the living lampreys should be ancestrally blood-feeders as the\ndentitional pattern represented by Ichthyomyzon species was always held as the primitive state\nfor living lampreys, see Potter & Hilliard, 1987; Hardisty 2015; Renaud & Cochran, 2019).\nAlso, the term “non-feeding forms” might be misleading to those unfamiliar with non-parasitic\nlampreys since they are only non-feeding after the onset of metamorphosis (Re: We have made it\nexplicit that here we refer to the non-feeding habit during the post-metamorphsis stage).\nLine 192-195 – It is not clear here if you are suggesting that Yanliaomyzon evolved a triphasic\nlife cycle or rather that it inherited it from the common ancestor that possessed a triphasic life cycle.\nI think the latter, but the wording could suggest the former.( Re: We have revised this part and\nmade it clear that ‘Yanliaomyzon species could likely have inherited such a life history strategy\nfrom the common ancestor’)\nLine 221 – The meaning of “which should be historical products of the enhancement of the feeding\nstructures” is not clear to me (Re: We have rephrased this part)\nLine 234-254\n– As with Line 188-192, this section seems rather simplistic, neither clearly explaining the “big\npicture” for a broad audience nor concisely and clearly describing what is novel here for a more\nexpert audience. (Re: We have tried to improve the expression of the co-evolutionary\ninteraction of the lampreys and their host (prey) victims, please check the revised MS. The\ntext in this part has been rephrased. Also, we have refreshed the text figures to help the readers\nunderstand our points)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily answered the majority of my concerns. There are still a few issues that\nI would like them to correct and new ones have cropped up. A copy editor will need to carefully go\nthrough the revised version because, for example, some spaces between words no longer exist (l.\n102), commas occur where they shouldn’t (l. 68), spaces need to be removed (l. 68), references\nshould start on a new line (l. 476).\nLines 66-67: Please remove “and posterior circumoral” because if there are no posterior disc teeth\nthere can’t be any posterior circumoral teeth either.\nThe authors use stage and phase indiscriminately. I use phase as a subset of stage (e.g., feeding\nphase of the adult stage). I have no problem if the authors choose to refer to a three-phase lamprey\nlife cycle as comprising the ammocoete, metamorphic, and adult, but they need to be consistent. It\nis confusing to readers if they call the three-phased lamprey life cycle, ammocoete, metamorphic,\nand adult stages.\nLine 84 and Fig. 2f: The two cusps flanking the two central cusps of the supraoral lamina in Y. occisor\ndo not appear smaller to me! They appear of similar size to the central cusps.\nLines 85 and 99: Remove “body very slender with” and “body relatively deep with the”,\nrespectively, because no measurement of the body depth was taken. Furthermore, remove “BD?”,\npresumably body depth, in line 685 of Supplementary Table 1 because this morphometric does not\nappear in the table.\nLine 99: Add the word slightly before “more than 40% of total body length.”\nLine 106: Add the common name Arctic lamprey before Lethenteron camtschaticum.\nLine 301: Lampreys do not use their oral disc to move sand, but use it to move stones to line the\nperiphery of a semi-circular redd. They move sand using their tail to create a central depression in\nthe redd into which the fertilized eggs fall into.\nClaude B. Renaud", "reviewer_2": "The authors have produced a well-rounded revision that addresses all the comments of the reviewers.\nI really appreciate the expanded discussion about the ecological factors behind the evolution of\nmodern lamprey, including the potential relationship between the loss of enamaloid in teleost scales\nand the evolution of parasitism. My main comment is that the writing in this new discussion section\nneeds additional polishing, as there are multiple grammatical errors (tense, extra spaces, etc). After\nthis is addressed, I believe this will be a valuable and surprising contribution to the literature on\nlamprey evolution and the evolution of fish biodiversity, worthy of publication in Nature\nCommunication.\nLauren Sallan", "reviewer_3": "GENERAL COMMENTS:\nThank you for your detailed and thoughtful responses to the excellent feedback you received from\nthe three reviewers and the substantial revisions that you made to the manuscript based on this\nfeedback. I think you have addressed most of the comments related to the manuscript’s content,\nalthough I bring up a few follow-up comments. I still think that a tighter, more concise discussion\nwould better convey and highlight the novel and exciting results of this paper, but I would not like to see\npublication of this work held up by several rounds of editing. However, I have pointed out some minor\ngrammatical errors or editorial issues that I think can be easily corrected.\nSPECIFIC COMMENTS REGARDING CONTENT\nLine 4 – By saying “the exemplar lineage,” does that exclude hagfishes? Would “one of two\nlineages” be more appropriate?\nLine 22 – What is meant by recent?\nLine 27 – As with Line 4, I’m not sure that lampreys are “the” representative lineage\nLine 31 – I would say: “in some cases, where they are non-native, they bring tremendous loss for\nfishery economy”. In their native ranges (i.e., everywhere but in the Laurentian Great Lakes),\nlampreys are part of a healthy ecosystem.\nLine 50 – Include scientific name on first mention?\nLine 60 – What classification are you following for Class Cyclostomi? Please indicate. As indicated\npreviously, recent classifications with which I am familiar (e.g., Nelson et al. 2016, Fishes of the\nWorld, 5th edition) place hagfishes and lampreys into separate classes (Myxini and Petromyzontida,\nrespectively). I recognize that not all authorities use the same classification, but please specify which\nrecent and accepted classification you are following.\nLine 253 – Although Yanliaomyzon occisor appears to have been anadromous, I’m not sure that I’m\nconvinced from this alone that “living lampreys were probably ancestrally anadromous.” For\nexample, I’m not sure that there might not have already been life history diversity in “pre-modern”\nlampreys. Also, it would be useful to specify why you suggest that anadromy was ancestral rather\nthan a purely marine ancestry (see comment on Line 326-332).\nLine 320-323 – I’m not sure that there is evidence that conodonts were the main competitor to\nlampreys (i.e., that they occupied the same ecological niche to the exclusion of many other jawless\nfishes). Furthermore, lampreys are no more closely related to conodonts than they are to other\nnon-cyclostome vertebrates (i.e., they last shared a common ancestor with conodonts at the same\ntime they last shared a common ancestor with the other non-cyclostome vertebrates).\nLine 326-332 – Interesting suggestion that the larger body size and hydromechanical advantage\nprovided by fin morphology permitted upstream migration and the evolution of anadromy rather\nthan vice versa. This is somewhat lost in what I think is still a longer, less focused discussion than\nit could/should be.\nReference 4 – It would be more useful if the specific chapter within this edited book is cited. It\nlooks like some references to this source come from the introductory chapter (Docker et al. 2015,\nIntroduction: A Surfeit of Lampreys, pp 1–34) while others (e.g., Line 302 come from Johnson et\nal. 2015, Reproductive Ecology of Lampreys, pp 265–303)\nReferences 7, 14 – What are the title of these chapters?\nEDITORIAL CORRECTIONS:\nLine 12, 38 – kin, not kins\nLine 15 – the Jurassic period (or the Jurassic)\nLine 29 – “sucking blood” and “gouging out tissues” still seems a bit colorful, but I’ll leave that to you\nand the editor\nLine 29, 54, and elsewhere – prey, not preys\nLine 31 – the aquatic ecosystem, the fishery economy\nLine 39, 41, and elsewhere – be consistent in spelling of ammocoete\nLine 252 – “which implies that this large lamprey would have been anadromous” would sound better\nLine 252 – phylogenetic\nLine 263 – with high hydromechanical efficiency\nLines 276, 336 – “epic odyssey” and “monstrous” still a bit colorful\nLine 277 – acting as\nLine 293, 304 – armour (not armours)\nLine 309 – “A food-driven tipping point for the prepared” sounds rather awkward\nLine 313-319 – a rather long, complicated sentence\nLine 319 – disappearance would sound better than vanishment\nLine 322 – went extinct\nLine 324 – delete “ ,” after with\nLine 367 – the southern and northern Pacific Ocean\nLine 375 – have led"}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have satisfactorily answered the majority of my concerns. There are still a few issues\nthat I would like them to correct and new ones have cropped up. A copy editor will need to\ncarefully go through the revised version because, for example, some spaces between words no\nlonger exist (l. 102)(Re: Corrected) , commas occur where they shouldn’t (l. 68) (Re:\nCorrected), spaces need to be removed (l. 68) (Re: Corrected), references should start on a new\nline (l. 476) (Re: Corrected, thank you very much).\nLines 66-67: Please remove “and posterior circumoral” because if there are no posterior disc teeth\nthere can’t be any posterior circumoral teeth either (Re: Revised accordingly).\nThe authors use stage and phase indiscriminately. I use phase as a subset of stage (e.g., feeding\nphase of the adult stage). I have no problem if the authors choose to refer to a three-phase lamprey\nlife cycle as comprising the ammocoete, metamorphic, and adult, but they need to be consistent. It\nis confusing to readers if they call the three-phased lamprey life cycle, ammocoete, metamorphic,\nand adult stages. (Re: Yes, indeed. We revised and made the usages consistent in the text)\nLine 84 and Fig. 2f: The two cusps flanking the two central cusps of the supraoral lamina in Y.\noccisor do not appear smaller to me! They appear of similar size to the central cusps. (Re: We\nrefer to the base of the central cusps and flanking cusps, with that of the former being larger\nthan the that of the latter)\nLines 85 and 99: Remove “body very slender with” and “body relatively deep with the”,\nrespectively, because no measurement of the body depth was taken (Re: Revised). Furthermore,\nremove “BD?”, presumably body depth, in line 685 of Supplementary Table 1 because this\nmorphometric does not appear in the table. (Re: Revised)\nLine 99: Add the word slightly before “more than 40% of total body length.” (Re: Added)\nLine 106: Add the common name Arctic lamprey before Lethenteron camtschaticum. (Re:\nAdded)\nLine 301: Lampreys do not use their oral disc to move sand, but use it to move stones to line the\nperiphery of a semi-circular redd. They move sand using their tail to create a central depression in\nthe redd into which the fertilized eggs fall into. (Re: Thank you very much for reminding these.\nWe have corrected the text)\nClaude B. Renaud\nReviewer #2 (Remarks to the Author):\nThe authors have produced a well-rounded revision that addresses all the comments of the\nreviewers. I really appreciate the expanded discussion about the ecological factors behind the\nevolution of modern lamprey, including the potential relationship between the loss of enamaloid in\nteleost scales and the evolution of parasitism. My main comment is that the writing in this new\ndiscussion section needs additional polishing, as there are multiple grammatical errors (tense,\nextra spaces, etc) (Re: Thank you. We have checked and revised accordingly). After this is\naddressed, I believe this will be a valuable and surprising contribution to the literature on lamprey\nevolution and the evolution of fish biodiversity, worthy of publication in Nature Communication.\nLauren Sallan\nReviewer #3 (Remarks to the Author):\nGENERAL COMMENTS:\nThank you for your detailed and thoughtful responses to the excellent feedback you received from\nthe three reviewers and the substantial revisions that you made to the manuscript based on this\nfeedback. I think you have addressed most of the comments related to the manuscript’s content,\nalthough I bring up a few follow-up comments. I still think that a tighter, more concise discussion\nwould better convey and highlight the novel and exciting results of this paper (Re: Thank you.\nWe have condensed some parts of the dicussion section, hopefully it is better now), but I\nwould not like to see publication of this work held up by several rounds of editing. However, I\nhave pointed out some minor grammatical errors or editorial issues that I think can be easily\ncorrected (Re: Indeed, we have checked and corrected).\nSPECIFIC COMMENTS REGARDING CONTENT\nLine 4 – By saying “the exemplar lineage,” does that exclude hagfishes? Would “one of two\nlineages” be more appropriate? (Re: Revised)\nLine 22 – What is meant by recent? (Re: Revised. We have specified it as ‘post-Cretaceous’)\nLine 27 – As with Line 4, I’m not sure that lampreys are “the” representative lineage (Re:\nRevised)\nLine 31 – I would say: “in some cases, where they are non-native, they bring tremendous loss for\nfishery economy”. In their native ranges (i.e., everywhere but in the Laurentian Great Lakes),\nlampreys are part of a healthy ecosystem. (Re: Thank you for reminding this. The text has been\nrevised accordingly)\nLine 50 – Include scientific name on first mention? (Re: Yes, the scientific name of the pouched\nlamprey is now included)\nLine 60 – What classification are you following for Class Cyclostomi? Please indicate. As\nindicated previously, recent classifications with which I am familiar (e.g., Nelson et al. 2016,\nFishes of the World, 5th edition) place hagfishes and lampreys into separate classes (Myxini and\nPetromyzontida, respectively). I recognize that not all authorities use the same classification, but\nplease specify which recent and accepted classification you are following. (Re: Revised. To avoid\nthe confusion, we deleted “Class Cyclostomi”, as the taxonomy of the crown-group\nvertebrates is beyond the scope of current study)\nLine 253 – Although Yanliaomyzon occisor appears to have been anadromous, I’m not sure that\nI’m convinced from this alone that “living lampreys were probably ancestrally anadromous.” For\nexample, I’m not sure that there might not have already been life history diversity in “pre-modern”\nlampreys. Also, it would be useful to specify why you suggest that anadromy was ancestral rather\nthan a purely marine ancestry (see comment on Line 326-332). (Re: Yes, you are right, this\npoint might be somehow lost in the lengthy discussion, which is now condensed. The\ninference of the anadromy of the node of the crown-group lampreys is based on the topology\n(Fig. 3) and the distribution of this trait in current phylogenetic framework. Given that\nYangliaomyzon occior and the earliest diverged crown lineage Geotria australis are all\nanadromous, the node of the crown-group (the theoretical ancestor of the crown-group\nlampreys) was inferred as an anadromous form. Indeed, we should do an ancestral trait\nreconstruction for this node, just like the reconstruction of the feeding (trait) habit.\nHowever, the emphasis of this study is the feeding biology, we did not do that for now.)\nLine 320-323 – I’m not sure that there is evidence that conodonts were the main competitor to\nlampreys (i.e., that they occupied the same ecological niche to the exclusion of many other jawless\nfishes). Furthermore, lampreys are no more closely related to conodonts than they are to other\nnon-cyclostome vertebrates (i.e., they last shared a common ancestor with conodonts at the same\ntime they last shared a common ancestor with the other non-cyclostome vertebrates). (Re: Indeed,\nthe phylogenetical status of the conodonts is disputed. To avoid the confusion, we have\ndeleted the ‘phylogenetically closely related’ in the text.\nAs for the ecological relationships between conodonts and lampreys, although Goudemand\net al. (2011), in addition to refs. (39, 43) cited in the original version of our MS, compared\nthe feeding apparatus of the conodonts and pouched lamprey Geotria australis and\nhighlighted the similarity of the feeding structures (and possibly the feeding habits) between\nthem, without any evidence of the presence of a suctorial oral disc in conodonts, admittedly\nit is not reasonable to assume these animals were ecological rivals in a given ecosystem.)\nLine 326-332 – Interesting suggestion that the larger body size and hydromechanical advantage\nprovided by fin morphology permitted upstream migration and the evolution of anadromy rather\nthan vice versa. This is somewhat lost in what I think is still a longer, less focused discussion than\nit could/should be. (Re: Thank you for your criticism. We have tried to revise and condense\nthis part. Please check the new version of the MS. The implications of large body size and\nthe hydromechanical advantages of the Jurassic lampreys were referred to in the first\nsection of the ‘Discussion’. Maybe we should have made a simulation and conducted\nhydrodynamical analyses to show the functional significance of lampreys’ evolving body size\nand fin morphology, but it seems to be too long to include these in current paper)\nReference 4 – It would be more useful if the specific chapter within this edited book is cited. It\nlooks like some references to this source come from the introductory chapter (Docker et al. 2015,\nIntroduction: A Surfeit of Lampreys, pp 1–34) while others (e.g., Line 302 come from Johnson et\nal. 2015, Reproductive Ecology of Lampreys, pp 265–303) (Re: Yes, indeed. Thank you for\nyour suggestion. Now the pages of the specific cited chapters are marked in the ref. list\naccording to the format of the journal (no title but the pages of the chapter).\nReferences 7, 14 – What are the title of these chapters? (Re: Ref. 7 refers to “Chapter 3 Post-\nmetamorphic Feeding in Lampreys”, according to the reference format of the journal, the\npages of the cited chapters were marked in ref. list; Ref. 14 refers to “Chapter 4 Life History\nEvolution in Lampreys: Alternative Migratory and Feeding Types”)"}]

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