Slimehead size through time: testing the temperature-size relationship in Late Cretaceous Trachichthyidae

As global temperature rises, fish are predicted to become smaller. Body size is a fundamental trait impacting many aspects of an animal’s life history and ecology, and understanding how it may respond to climate change in particular fish groups, especially commercial or keystone species, is critical. The slimeheads (Family Trachichthyidae) include several commercially important species, but because they are deep-dwelling, long-lived fish, which reproduce slowly, directly testing the temperature-size relationship in this family is challenging. Fortunately, the Trachichthyidae have a long evolutionary history beginning in the Cretaceous and their fossil record provides empirical data on the response of this family to past climate change events. In this study, we leveraged the extensive fossil record of the Late Cretaceous trachichthyid genus Hoplopteryx from the British Chalk Group of southern England, United Kingdom to test whether its size declined at higher temperatures. Standard Lengths were measured from complete individuals and estimated from partial remains. Seawater palaeotemperature estimates were derived from oxygen stable isotope values (δ 18 O) of the bulk chalk rock surrounding the fossils using standard techniques and assumptions. Individual fish ranged from 56.3 to 253.5 mm in length, and measured seawater temperature estimates ranged from 19.5 to 27.1 °C. We recorded a significant negative correlation between estimated seawater temperature and Standard Length in the most common species Hoplopteryx lewesiensis, supporting the prediction that higher temperatures lead to smaller body size. In addition, we found a significant negative correlation between the lengths of H. lewesiensis and carbon stable isotope values (δ 13 C) of the chalk matrix, suggesting that other environmental factors such as primary productivity and/or the burial of organic matter may also have affected body size. Slimehead size through time: testing the temperature-size relationship in Late Cretaceous Trachichthyidae Chloe V. Griffiths 1,2, James D. Witts 1, Julie C. S. Brown 2, Emma L. Bernard 1, Richard J. Twitchett 1 1 Natural History Museum, Cromwell Road, London, SW7 5BD, United Kingdom 2 University College London, Gower Street, London, WC1E 6BT, United Kingdom Address information of the corresponding author: Chloe Griffiths Natural History Museum, Cromwell Road, South Kensington, London, United Kingdom, SW7 5BD

As global temperature rises, fish are predicted to become smaller. Body size is a fundamental trait impacting many aspects of an animal’s life history and ecology, and understanding how it may respond to climate change in particular fish groups, especially commercial or keystone species, is critical. The slimeheads (Family Trachichthyidae) include several commercially important species, but because they are deep-dwelling, long-lived fish, which reproduce slowly, directly testing the temperature-size relationship in this family is challenging. Fortunately, the Trachichthyidae have a long evolutionary history beginning in the Cretaceous and their fossil record provides empirical data on the response of this family to past climate change events. In this study, we leveraged the extensive fossil record of the Late Cretaceous trachichthyid genus Hoplopteryx from the British Chalk Group of southern England, United Kingdom to test whether its size declined at higher temperatures. Standard Lengths were measured from complete individuals and estimated from partial remains. Seawater palaeotemperature estimates were derived from oxygen stable isotope values (𝛿 18 O) of the bulk chalk rock surrounding the fossils using standard techniques and assumptions. Individual fish ranged from 56.3 to 253.5 mm in length, and measured seawater temperature estimates ranged from 19.5 to 27.1 ℃. We recorded a significant negative correlation between estimated seawater temperature and Standard Length in the most common species Hoplopteryx lewesiensis, supporting the prediction that higher temperatures lead to smaller body size. In addition, we found a significant negative correlation between the lengths of H. lewesiensis and carbon stable isotope values (𝛿 13 C) of the chalk matrix, suggesting that other environmental factors such as primary productivity and/or the burial of organic matter may also have affected body size.

(4-6) Fish, body size, climate change, fossil, temperature

Body size is a fundamental trait which affects many aspects of an animal’s life history, ecology and evolution (Peters, 1983). It is also a trait that responds to external, environmental factors, such as temperature, as outlined by the ecogeographical rules of Bergmann (1847) and James (1970), and the temperature-size rule (Atkinson, 1994), where warmer waters tend to be inhabited by smaller-sized species and populations (Daufresne et al., 2009). This relationship between temperature and body size has been tested in many living fish, for example, van Rijn et al. (2017) showed that maximum annual sea surface temperature is the best predictor of body size in 74 species. Based on this relationship, it is predicted that as global temperatures increase with current climate change, many species will become smaller in response (Forster et al., 2012; Millien et al., 2006; Sheridan and Bickford, 2011). Fish are not expected to respond to temperature alone, but to a range of related factors such as dissolved oxygen content, pH and productivity (Mora et al., 2013). For example, as seawater temperature increases, the dissolved oxygen content decreases and fish growth rate slows (Pörtner and Knust, 2007). By 2050 it is predicted that fish maximum body weight will have shrunk by up to 24% (Cheung et al., 2013). There is evidence that larger-bodied, commercially important species are disproportionately decreasing in size and abundance compared to smaller species (Baudron et al., 2014; Genner et al., 2010; Todd et al., 2008), although as fishing itself is a size-selective activity this may not be solely due to climate change. Nonetheless, body size changes resulting from temperature changes may have negative impacts on fish at individual, population, and ecosystem levels, including reduced fecundity, and reduced offspring survival (Ahti et al., 2020). These negative impacts could result in population declines for species that grow and reproduce slowly. Further research is necessary to understand how climate change may affect different fish species, particularly those that are more vulnerable such as those with slow life histories. Trachichthyidae, also known as ‘slimeheads’, are a family of commercially exploited fish found in the Atlantic, Pacific and Indian oceans (Nelson et al., 2016). They typically occupy depths ranging from 100 to 1500 metres, depending on species and maturity (Lorance et al., 2002; Madurell and Cartes, 2005), where they are sluggish predators feeding on crustaceans, other fish and squid (Bulman and Koslow, 1992; Madurell and Cartes, 2005). Trachichthyidae have high metabolic costs (Bulman and Koslow, 1992), grow slowly, mature late in life (some species do not reach reproductive maturity until 20 years old), and display low fecundity (D’onghia et al., 1998; Mace et al., 1990). They are extremely long-lived, with individuals of some species documented to reach 50 to 100 years of age (Lorance et al., 2002; Mace et al., 1990). It is extremely difficult to observe biological changes in Trachichthyidae over short study periods, due to their deep-sea habit and slow life history (Clark et al., 2000) and hitherto it has not been possible to experimentally test the temperature-size relationship in these fish. Only one species, Hoplostethus atlanticus, has been subject to any temperature-related studies. Using otoliths to estimate growth rates, and the Mg/Ca ratio of deep-sea corals as a temperature proxy, Thresher et al. (2007) inferred a positive relationship between the growth rate of juvenile H. atlanticus and temperature over the past 200 years. How temperature may relate to adult body size was not examined. In this study, we take a different approach and utilise the fossil record as an alternative source of data to test the size-temperature relationship in the Trachichthyidae. Originating in the Cretaceous (Berg, 1958; Patterson, 1964), the Trachichthyidae have a long evolutionary history and have experienced many past episodes of global climate change. The fossil record of these events can be viewed as a series of ‘natural experiments’ (Jablonski, 2004), that provide an opportunity to test the relationship between palaeotemperature and body size in extinct members of extant clades. This may contribute to better prediction of the responses of living species to current climate change (Millien et al., 2006). We focused on the extinct trachichthyid genus Hoplopteryx, which ranges from the Cenomanian to Campanian stage of the Late Cretaceous (ca. 100.5 to 72.1 million years ago) (Cohen et al., 2013; Patterson, 1964). Fossils of Hoplopteryx are relatively common in the British Chalk Group of southern England, which was originally deposited as a coccolith ooze on the floor of an epeiric sea no deeper than 200-500 m (Bell et al., 1999). Chalk fossils have been collected for over 200 years, and specimens of Hoplopteryx are housed in many United Kingdom (UK) museum collections. Furthermore, the Chalk Group has been the subject of palaeoclimatological studies for more than three decades as it records several significant global warming and cooling events (Jarvis et al., 2006; Jenkyns et al., 1994), and geochemical methods are well established for estimating trends in Chalk Sea water temperature from oxygen stable isotope values (𝛿 18 O; Anderson and Arthur, 1983; Jenkyns et al., 1994). This study aimed to provide the first test of the size-temperature relationship in the Trachichthyidae by analysing size change in the extinct, Late Cretaceous genus Hoplopteryx Hoplopteryx from the British Chalk Group of southern England. Fossil fish body size data were compared to estimates of seawater palaeotemperature inferred from the 𝛿 18 O of the bulk chalk matrix surrounding individual fossils. To explore other potential palaeoenvironmental influences on body size, we also investigated the relationship between size and carbon stable isotope values (𝛿 13 C).

Fossilised specimens of Hoplopteryx were sourced from the Natural History Museum, London (NHMUK), the British Geological Survey, Keyworth (BGS), the Grant Museum of Zoology, London (GMZ) and the Sedgwick Museum of Earth Sciences, Cambridge (CMZ). In total, 250 Hoplopteryx specimens were surveyed. Ten specimens labelled Hoplopteryx superbus were excluded from this study, as it is uncertain whether this species belongs to Hoplopteryx or to Caproberyx (Patterson, 1964). Of the remaining specimens, a further 26 were not sufficiently well articulated or preserved to be analysed further, leaving a total dataset of 214 specimens. All specimens were originally collected from inland and coastal exposures of the British Chalk Group of southern England, UK (Figure S1). Seven morphological parameters (Fig. 1; Table 1) were measured using Mitutoyo digital callipers accurate to 0.01 mm. All specimens were measured once, and a random subset of 23 were re-measured to verify internal consistency and reproducibility. All repeats were within ± 2% (Table S1). For these repeat-measured specimens the mean values between the two measurements were used in all further analyses. Standard Length (SL) was chosen to represent body size in this study. Three fish were preserved with their bodies in a curved or twisted position. In order to measure SL in those specimens, a piece of string was used which was then straightened out and measured with callipers. The process was repeated three times, and a mean SL was calculated. Morphometric analyses Most fossils were incomplete or still retained adhering chalk matrix which prevented direct measurement of the SL. To include these specimens in our study, morphometric analyses were undertaken to determine whether alternative morphological parameters could provide robust estimates of SL in partial specimens, and, if so, which parameter(s) gave the most precise and accurate estimate. Six skeletal parameters were tested: Head Length, Head Height, Jaw Length, Orbit Diameter, Trunk Length and Vertebrae Width (Table 1). These parameters were measured in the most complete specimens of Hoplopteryx spp. ( n = 46) and linear regressions between each parameter and SL were generated. The robustness of each morphological parameter for predicting SL was determined by the R 2, F -statistic and residual standard error of each linear regression (Engelman, 2023; Grouard et al., 2019). Measurements of the best-performing parameters were then utilised to estimate SL in partial specimens, using the equation: \(\text{Standard\ Length\ }\left(\text{SL}\right)=\left(slope\ \times\ parameter\right)+intercept\)(1) Each regression analysis was tested using the correlation coefficients to compare estimated SL to measured SL in complete specimens. Carbon and oxygen stable isotope analysis Samples of chalk matrix from 54 NHMUK Hoplopteryx specimens were selected on completeness and to encompass as large a range of recorded body size as possible. Bulk chalk matrix was weighed (650–750 μg each) and transferred to a 12 mL Labco vial that was then screw-capped with butyl rubber septa and flushed with ultra-pure helium. Samples were reacted with 100 µL phosphoric acid (density 1.92 g mL -1 ). Stable isotope analysis was carried out on a DELTA V Advantage continuous flow isotope ratio mass spectrometer (IRMS) linked to a GasBench II via a ConFlo IV with a GC PAL autosampler (Thermo Fisher Scientific, Germany) at the Bloomsbury Environmental Stable Isotope Facility at University College London in London, UK. IRMS linearity was always < 0.06 ‰ volt -1 for a defined span of carbon dioxide (CO 2 ) voltage, and the standard deviation of values of δ 13 C and δ 18 O from ten peaks of CO 2 working gas was always < 0.06 ‰. Sample CO 2 evolved from acidification was purged from vials through a double-hole sampling needle into a helium carrier. After drying, the CO 2 was separated from residual gases by a chromatographic column (PoraPLOT Q) at 70°C. Repetitive loop injection allowed for the transfer of ten headspace CO 2 samples per vial. Values of 𝛿 13 C and 𝛿 18 O were derived relative to the CO 2 working gas introduced directly into the IRMS and were corrected for 17 O (Santrock et al., 1985) by Isodat software (version 3.0.95.23; Thermo Fisher Scientific, Germany). Stable isotope ratios, expressed as delta values (δ) in per mil units (‰; Brand et al., 2014), represent the ratio of heavy to light isotopes within a sample (R sample ) relative to the ratio in an international standard (R standard ): δ = (R sample / R standard )-1 (Coplen, 2011). Sample CO 2 peaks number two to ten (excluding sample peak one) were used to calculate mean isotopic delta values. All values of δ 13 C and δ 18 O are reported relative to the international reference standard Vienna Pee Dee Belemnite (VPDB). International Atomic Energy Agency (IAEA) reference materials (RMs) (Tables S2 and S3) were analysed in duplicate at the start and end of each sample batch. A calcium carbonate laboratory standard was analysed at regular intervals throughout each sample batch. Isotopic values from RMs and laboratory standard were used to assess drift and linearity. No drift or linearity corrections were applied. Values were checked for outliers. No oxygen-isotope acid fractionation factor was applied because the carbonate present in the samples was expected to be calcite. The acid fractionation factor would be identical for samples and reference materials and its value would therefore cancel out (Kim et al. 2015). Data from IAEA-603 and IAEA-610 were used to scale-correct all delta values (Paul et al., 2007). Accuracy and precision data from the analysis of RMs and laboratory standard calcium carbonate are reported in Tables S2 and S3. Palaeotemperature reconstruction The Upper Cretaceous Chalk of the UK and northwest Europe formed from carbonate ooze which originally accumulated on the seafloor as marine snow and is composed primarily of the microscopic fossil skeletons of calcareous plankton, mainly coccolithophores (nannofossils), with minor components from other biotic and abiotic sources (Fabricius, 2007; Püttmann and Mutterlose, 2021). Therefore, the oxygen and carbon isotope compositions of the bulk chalk can provide insight into the environmental conditions in the upper parts of of the water column, as experienced by the nannofossils preserved within it (Wefer and Berger, 1991). Following the approach of Jenkyns et al. (1994), the relative seawater palaeotemperature of each chalk sample was estimated using the equation: \(T\left({{}^{\circ}}C\right)=16.0-4.14\left(\delta c-\ \delta w\right)+\ 0.13\left(\delta c-\ \delta w\right)^{2}\ \), (2) in which 𝛿c is the measured oxygen isotope composition of the bulk chalk matrix and 𝛿w is the oxygen isotope composition of seawater (Anderson and Arthur, 1983). The oxygen isotope composition of Chalk Sea seawater is unknown. Therefore, in common with many other palaeoclimate studies a seawater δ 18 O SMOW (Standard Mean Ocean Water) of −1‰ was used, based on the assumption that this records the average value for an ice-free, greenhouse Late Cretaceous world (Wilkin, 2021). Statistical analyses The variances of SL, 𝛿 18 O, estimated seawater temperature, and 𝛿 13 C values were homogenous . However, as SL, 𝛿 18 O and seawater temperature values were not normally distributed, the non-parametric Spearman’s ρ was used to test the strength and significance of the correlation between parameters. Relationships between SL and 𝛿 18 O, seawater temperature and 𝛿 13 C were analysed at both the genus-level (i.e the entire dataset) and at the species-level where sufficient specimens (minimum n = 20) were present.

Of the 214 specimens measured in this study, only 46 had directly measurable SLs, which ranged from 58.8 mm to 221.2 mm (Table S4). Morphometric analyses of those 46 specimens showed that each of the six morphological parameters tested increased with SL (Figure 2). Following regression analyses, Orbit Diameter, Vertebrae Width and Jaw Length were discounted as predictors of SL as the R 2 was lower than 80%, the F -statistics were relatively low and the residual error was relatively high (Table 2), indicating that these parameters poorly explained the variability in SL. Although Trunk Length R 2 was higher than 80%, a relatively high F -statistic and relatively low residual error, the sample size was much smaller than that of the other parameters ( n < 20). Therefore, Trunk Length was also discounted as a predictor of SL. Head Length and Head Height demonstrated the highest R 2 values, highest F -statistics, and the lowest residual error, indicating that these two parameters best explain the variability in SL (Table 2). Therefore, coefficients from linear regressions using the predictor parameters Head Length (Slope = 2.92, Intercept = 4.28) and Head Height (Slope = 2.12, Intercept = 10.11) were inserted into Equation 1 to estimate SL for incomplete specimens. In specimens where both Head Length and Head Height were available, the mean of the individual SL estimates from both Head Length and Head Height was used as the final estimate of SL. Of the 54 samples analysed geochemically, 26 were associated with specimens of fish where SL was directly measured and 28 where SL was estimated from direct measurements of Head Length, Head Height or a mean of both. Forty-seven samples were from specimens of H. lewesiensis, with the remaining 7 either unassigned to species or assigned to other species. Measured 𝛿 18 O values ranged from -1.9 to -3.6‰, which equated to seawater temperature estimates of 19.5 to 27.1°C respectively (Table 3). For H. lewesiensis, a significant inverse relationship between SL and estimated seawater temperatures is recorded ( r (45) = -.35, p = .015), indicating that sizes are smaller at higher estimated temperatures (Figure 3). There were insufficient specimens to test this relationship in other Hoplopteryx species separately. For the whole Hoplopteryx spp. dataset ( n = 54), SL was inversely correlated to estimated temperature, but this relationship was not significant ( r (52) = -.13, p = .34). Measured 𝛿 13 C values ranged from 1.3 to 4.2‰ across the 54 samples, (Table 3). There was a strong, significant negative correlation between SL and 𝛿 13 C in both H. lewesiensis ( r (45) = -.54, p = <.001) and in Hoplopteryx spp. ( r (52) = -.45, p = <.001) (Figure 4). Notably, there was no significant correlation between 𝛿 13 C and 𝛿 18 O in the chalk matrix samples ( R 2 = <.01) (Figure S2), suggesting the two palaeoenvironmental proxies are independent of each other. Furthermore, the lack of correlation between carbon and oxygen isotope values is an indicator of weak pore fluid-rock interaction, and therefore minimal diagenetic alteration to isotopic signatures (Huber et al., 2024). Body size and seawater temperature By combining morphometric measurements of fossil specimens with geochemical analysis of their surrounding chalk matrix, our study provides the first test of the size-temperature relationship in an extinct member of the extant and commercially important deep-sea fish family Trachichthyidae. We found a significant positive correlation between the Standard Length of individuals of the species Hoplopteryx lewesiensis, which is the most common trachichthyid species from the Upper Cretaceous British Chalk Group, and the 𝛿 18 O values of the chalk matrix surrounding each specimen. When 𝛿 18 O values were converted to estimates of seawater temperature, following a well-established approach and using standard assumptions, our results show a significant negative correlation between the Standard Length of H. lewesiensis and seawater temperature; i.e. body size decreased with increasing seawater palaeotemperature. These results are consistent with studies of similarly sized extant fish, which show that higher temperatures have led to smaller body size (Audzijonyte et al., 2020). They provide further support for the prediction that marine animals in general, and fish in particular, will decline in body size with current and future ocean warming owing to reduction in dissolved oxygen concentrations (e.g. Sheridan and Bickford, 2011; Cheung et al., 2013). Although it is possible that the Trachichthyidae may have evolved greater resilience to temperature change since the Late Cretaceous, given the limited data available for living slimeheads (Clark et al., 2000; Thresher et al., 2007) , our results provide an insight into how current climate warming may impact the body sizes of living members of this family. We did not find a significant relationship between estimated seawater temperature and body size in our genus-level analysis, which we attribute to the confounding effect of inter-species variation in body size in the different Hoplopteryx species (Patterson, 1964). Furthermore, a number of specimens within our genus-level database were unable to be assigned to individual species, and it is possible that they may represent juveniles rather than adults (see discussion below). For those specimens that were assigned to other species, (e.g. to H. simus, H. macrocanthus ), there were insufficient numbers to undertake robust species-level statistical analyses. Body size and 𝛿 13 C A significant negative correlation between the Standard Length and the δ 13 C values of the surrounding chalk matrix was found for both the genus Hoplopteryx and for the species H. lewesiensis, with the smallest fish being associated with the most positive δ 13 C values. The interpretation of these results is not straightforward because there are many interacting biotic and abiotic factors which can affect the 𝛿 13 C signature of marine carbonates such as chalk. The primary controls on 𝛿 13 C in chalk are widely considered to be primary productivity and burial of organic matter in seafloor sediments (Jarvis et al., 2002, 2006; Mitchell et al., 1996). Carbon isotope fractionation during marine photosynthesis leads to organic matter enriched in 12 C and the remaining carbon pool in the surrounding water, from which marine organisms precipitate their skeletal carbonates and, therefore becomes relatively enriched in 13 C (Jarvis et al., 2002). The burial of this 13 C-depleted organic matter in seafloor sediments further enhances this process by removing relatively more 12 C from the system. Thus, during times of enhanced productivity and/or greater burial of organic matter, the 𝛿 13 C of marine carbonates is expected to be more positive (e.g. Mitchell et al., 1996). Our results, therefore, indicate that Hoplopteryx became smaller with an increase in productivity and/or burial of organic matter. Interpreting these results in terms of changes in productivity is problematic. Although we have no direct evidence of the diet of Hoplopteryx, it might be expected that greater primary productivity would have increased available food supply at higher trophic levels, which in turn would lead to an increase in Hoplopteryx body size (e.g. Mora et al., 2013 and references therein) . Unless, perhaps, Hoplopteryx was adapted to more oligotrophic conditions, which have been inferred for the offshore environments of the UK chalk seas (Püttmann and Mutterlose, 2021), and higher productivity caused a negative impact on body size. Our results are easier to interpret, however, if burial of organic matter is the most significant factor controlling the 𝛿 13 C of the bulk chalk. Increasing organic matter burial could be achieved by increasing the area of seafloor available, for example at times of higher global sea-level (Mitchell et al., 1996), or by reducing the dissolved oxygen content at or near the seafloor, enhancing preservation potential (Jarvis et al., 2002). The latter could explain why Hoplopteryx size decreases as 𝛿 13 C increases, as reduced oxygen availability is known to limit fish growth and size (e.g. Cheung et al., 2013; Pörtner and Knust, 2007; Sheridan and Bickford, 2011). Standard Length increases through ontogeny and in some fish, there is also a substantial size difference between the two sexes. It is, unfortunately, often impossible to determine either the sex or ontogenetic stage of a fossilised individual, unless ontogenetic differences or sexual dimorphism are clearly recorded in the preserved skeletal tissues. Here we consider the extent to which either factor could have biased these results. In extant Trachichthyidae, there is evidence of sexual dimorphism in some species, but not all (D’onghia et al., 1998; Dunn and Forman, 2011; Elliott et al., 1995; Shimizu, 1977). Even in species where it may occur, the size difference between sexes is relatively small (D’onghia et al., 1998; Elliott et al., 1995; Tracey, 1999). Determining the sex of a fossil fish from partial skeletal remains is challenging at best and, in common with nearly all palaeontological studies, we were not able to do this for our specimens. We note, however, that the size distribution of our dataset is unimodal (Figs. S3 and S4), and so even if Hoplopteryx is shown to have size-related sexual dimorphism, it is unlikely to have biased our data or caused the significant correlations between Standard Length and chalk 𝛿 18 O values/seawater temperature or chalk 𝛿 13 C values. The possible impact of ontogeny on our results is more difficult to determine, as methods to determine ontogeny in fish from skeletal remains are limited. Biological age determination using otoliths is the most reliable method of age determination in fishes (Khan et al., 2013). However, otoliths have not been reported from any British Chalk Group fish taxon, probably owing to early diagenetic loss of aragonite (Friedman et al., 2016), so we could not use this technique. Instead, we attempted to assess the biological age of individual specimens by counting the circuli and annuli preserved in their scales. Unfortunately, very few individuals ( n = 11) preserved visible, measurable circuli, and therefore the correlation between size and age was inconclusive. An alternative approach may be to use computed tomography to image the scales (Thomson and McCune, 1984), but this was not possible during our study. In the absence of ontogenetic data, we therefore assume that the randomly selected subset of individuals chosen for geochemical analysis all represent adults.

Our study is the first to demonstrate a significant relationship between seawater palaeotemperature and the body size of an extinct species of Trachichthyidae. We show a significant negative correlation between estimated seawater temperature, inferred from oxygen isotope analysis, and body size in the Late Cretaceous species Hoplopteryx lewesiensis from the British Chalk Group . Additionally, a significant negative correlation was found between Hoplopteryx body size and carbon isotope values (𝛿 13 C) of the surrounding chalk, indicating that other abiotic factors also influenced body size in this group of fish through the Late Cretaceous. Our study demonstrates the importance of utilising the fossil record as an alternative source of data to test the size-temperature relationship in marine animals such as deep-sea fish. LITERATURE CITED Ahti, P.A., Kuparinen, A. and Uusi-Heikkilä, S. 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TABLES Table 1: Definitions of the seven morphological parameters measured in this study . | Head Length | Length from the tip of the snout to the anterior of the operculum | | Head Height | Length from the most dorsal to the most ventral point of the head, perpendicular to head length | | Jaw Length | Length from the most anterior to the most posterior point of the visible jawbone(s) | | Orbit Diameter | Length of the eye socket from the most anterior to the most posterior point | | Trunk Length | Length from the most anterior point behind the head, to the point just before any preserved a-l fin rays | | Vertebrae Width | Length of the most anterior visible vertebra, from its most anterior to most posterior point | | Standard Length (SL) | Length from the tip of the snout to the base of the tail | Table 2: Results of regression analyses of each morphological parameter. | Head Length | 39 | 0.89 | 294.4 | 13.69 on 38 df. | | Head Height | 33 | 0.85 | 185.8 | 14.53 on 32 df. | | Trunk Length | 15 | 0.91 | 142.3 | 13.28 on 14 df. | | Vertebrae Width | 32 | 0.78 | 110.3 | 18.94 on 31 df. | | Jaw Length | 35 | 0.74 | 96.35 | 18.54 on 34 df. | | Orbit Diameter | 31 | 0.61 | 46.23 | 23.70 on 30 df. | Table 3: Geochemical and Standard Length (SL) data collected from Hoplopteryx spp. specimens at the Natural History Museum, UK. Estimated SL are in bold. Italicised SL are the mean of two measurements. Minimum and maximum estimated or measured SL are listed where applicable. | PV OR 36917(a) | lewesiensis | -2.82 | 23.7 | 2.29 | 61.6 | 61.4 | 61.9 | | PV P 5423 | lewesiensis | -3.3 | 25.9 | 3.02 | 80.6 | 76.45 | 84.7 | | PV OR 25827 | lewesiensis | -2.93 | 24.2 | 2.09 | 82.2 | 81.4 | 83.1 | | PV OR 41993 | ? | -1.86 | 19.5 | 2.02 | 83.2 | - | - | | PV P 10222 | simus | -2.53 | 22.4 | 1.62 | 86.7 | 85.9 | 87.5 | | PV OR 4012 | lewesiensis | -3.38 | 26.3 | 2.32 | 90.1 | - | - | | PV P 6464 | lewesiensis | -2.99 | 24.5 | 2.99 | 96 | 92.7 | 99.4 | | PV OR 4008 | lewesiensis | -3.31 | 25.9 | 2.41 | 107.5 | 107.5 | 107.6 | | PV OR 4026 | lewesiensis | -3.28 | 25.8 | 2.4 | 113.4 | 111.9 | 114.8 | | PV OR 33230 | macrocanthus | -2.2 | 21 | 1.91 | 113.5 | - | - | | PV OR 49038 | lewesiensis | -2.89 | 24 | 2.24 | 113.7 | - | - | | PV P 1948(d) | lewesiensis | -3.04 | 24.7 | 3.01 | 116.1 | - | - | | PV OR 25912 | lewesiensis | -3.3 | 25.9 | 1.38 | 117.2 | 105.5 | 129.0 | | PV P 1948(a) (1) | lewesiensis | -3.12 | 25.1 | 1.93 | 118.6 | 118.0 | 119.1 | | PV OR 4011 | lewesiensis | -3.22 | 25.5 | 2.28 | 127.2 | - | - | | PV OR 25863 | lewesiensis | -3.09 | 24.9 | 2.39 | 130.2 | 124.2 | 136.1 | | PV OR 49041(1) | lewesiensis | -2.97 | 24.4 | 1.58 | 131.9 | - | - | | PV OR 49888 | lewesiensis | -3.36 | 26.2 | 2.88 | 132 | 118.4 | 145.6 | | PV OR 49867(1) | lewesiensis | -3.27 | 25.7 | 1.92 | 134.8 | - | - | | PV P 5420 | lewesiensis | -2.69 | 23.1 | 3.14 | 141 | 137.4 | 144.6 | | PV P 4842 | lewesiensis | -2.98 | 24.4 | 2.21 | 143.3 | - | - | | PV OR 4019 | lewesiensis | -2.93 | 24.2 | 1.53 | 146.7 | 142.5 | 151.0 | | PV OR 4014 | lewesiensis | -2.33 | 21.5 | 1.94 | 149.4 | 148.8 | 150.1 | | PV OR 4016 | lewesiensis | -2.98 | 24.4 | 2.83 | 151.5 | 149.1 | 154.0 | | PV OR 49870 | lewesiensis | -3.38 | 26.3 | 2.35 | 151.8 | - | - | | PV OR 41104 | gephyrognathus | -2.89 | 24 | 4.24 | 158.2 | 145.8 | 170.6 | | PV OR 25781 | lewesiensis | -2.63 | 22.9 | 3.01 | 158.2 | 157.7 | 158.6 | | PV OR 4106 | lewesiensis | -2.8 | 23.6 | 2.12 | 158.4 | 149.8 | 167.1 | | PV OR 25841 | lewesiensis | -3.11 | 25 | 1.91 | 162.4 | - | - | | PV P 5690 | lewesiensis | -2.85 | 23.8 | 2 | 162.5 | 159.7 | 165.3 | | PV P 7189 | lewesiensis | -3.56 | 27.1 | 1.92 | 162.5 | 154.3 | 170.7 | | PV OR 41105(a) | lewesiensis | -2.67 | 23 | 2.03 | 164.2 | 161.0 | 167.5 | | PV OR 4015 | lewesiensis | -2.24 | 21.2 | 1.93 | 165.8 | 151.8 | 179.8 | | PV OR 4109 | lewesiensis | -2.86 | 23.9 | 1.3 | 166.8 | - | - | | PV P 5692 | lewesiensis | -3.24 | 25.6 | 1.71 | 166.9 | 166.0 | 167.8 | | PV P 73798 | lewesiensis | -3.07 | 24.8 | 2.98 | 167.7 | - | - | | PV P 5693 | lewesiensis | -3.17 | 25.3 | 1.7 | 169.4 | 158.5 | 180.4 | | PV P 1948(b) | lewesiensis | -2.69 | 23.1 | 1.75 | 171.1 | 170.9 | 171.4 | | PV OR 4021a | lewesiensis | -3.32 | 26 | 1.96 | 172.4 | 164.2 | 180.6 | | PV OR 79 | lewesiensis | -2.37 | 21.7 | 1.98 | 172.4 | 171.4 | 173.4 | | PV OR 28392 | ? | -3.21 | 25.5 | 1.41 | 177.5 | 166.7 | 188.3 | | PV OR 49037 | lewesiensis | -2.67 | 23 | 2.02 | 179.9 | 174.2 | 185.5 | | PV P 5694 | lewesiensis | -2.96 | 24.3 | 1.92 | 180.7 | - | - | | PV P 5688 | lewesiensis | -3.12 | 25.1 | 1.71 | 180.9 | 178.5 | 183.3 | | PV P 4297 | sp | -2.97 | 24.4 | 1.98 | 184.2 | 177.5 | 190.9 | | PV P 5689 | lewesiensis | -3.21 | 25.5 | 1.73 | 185.6 | 181.3 | 190.0 | | PV P 51289 | lewesiensis | -2.83 | 23.8 | 1.96 | 190.2 | - | - | | PV OR 49862 | lewesiensis | -2.77 | 23.5 | 1.96 | 195.8 | 176.9 | 214.7 | | PV P 5687 | lewesiensis | -3.3 | 25.9 | 1.62 | 198.0 | 196.09 | 199.95 | | PV P 9909 | lewesiensis | -2.75 | 23.4 | 1.27 | 204.1 | - | - | | PV OR 49863 | lewesiensis | -2.53 | 22.4 | 1.47 | 205.6 | 188.4 | 222.9 | | PV OR 35712 | lewesiensis | -2.91 | 24.1 | 2.1 | 208.3 | 208.2 | 208.5 | | PV OR 49043 | lewesiensis | -2.53 | 22.4 | 1.63 | 221.2 | 220.6 | 221.8 | | PV OR 4239 | ? | -3.17 | 25.3 | 1.93 | 222.8 | 197.5 | 248.1 | FIGURE LEGENDS Figure 1: Hoplopteryx lewesiensis specimen (NHMUK PV P 51289) labelled with the seven morphological parameters measured in this study (definitions in Table 1). Figure 2: The relationship between Standard Length (SL) and each morphometric parameter in Hoplopteryx spp. Each dot represents one specimen. Colours represent different morphological parameters, as shown in the key. A regression line (y~x) has been fitted to each parameter dataset. Sample sizes are shown on the plot. Figure 3: The relationship between 𝛿 18 O-derived seawater temperature estimates and Standard Length (SL) in (A) Hoplopteryx spp. and (B) Hoplopteryx lewesiensis . Estimated points show a mean from Head Length and Head Height SL estimates. Measured points either show the true SL measurement or a mean of two repeats. The error bars on estimated SL points show the smallest and largest estimates. No error bars are shown on measured SL points as the smallest and largest values of repeat measurements were smaller than the size of the symbol. Sample sizes are shown on each plot. A regression line (y~x) is added to each plot and the grey-shaded areas represent a 95% confidence interval. Figure 4: The relationship between 𝛿 13 C and Standard Length (SL) in (A) Hoplopteryx spp. and (B) Hoplopteryx lewesiensis . Estimated points show a mean from Head Length and Head Height SL estimates. Measured points either show the true SL measurement or a mean of two repeats. The error bars on estimated SL points show the smallest and largest estimates. No error bars are shown on measured SL points as the smallest and largest values of repeat measurements were smaller than the size of the symbol. Sample sizes are shown on each plot. A regression line (y~x) is added to each plot and the grey-shaded areas represent a 95% confidence interval. DATA ACCESSIBILITY STATEMENT The authors confirm that the data supporting the findings of this study are available within the article and its Supporting Information. COMPETING INTERESTS STATEMENT None declared. AUTHOR CONTRIBUTIONS SECTION Conceptualization: ALL; Data Curation: CVG, ELB; Formal Analysis: CVG, JCSB; Funding Acquisition: RJT; Investigation: CVG, JCSB; Methodology: ALL; Writing – Original Draft Preparation: CG; Writing – Review & Editing: ALL

This project was supported by CVG’s MRes project bench fees, and by the Natural Environment Research Council (NERC)-funded Chalk Sea Ecosystems (ChaSE) project NE/X015300/1 led by RJT. Kim Chandler (NHMUK) is thanked for providing curatorial support. Hannah Cornish (Grant Museum of Zoology), Matt Riley (Sedgwick Museum of Earth Sciences) and Paul Shepherd (British Geological Survey), are thanked for assistance in accessing collections in their care. Supplementary Material File (supporting_information.docx) - Download - 530.87 KB Information & Authors Information Version history Peer review timeline Published Ecology and Evolution Version of Record25 Sep 2025Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection

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