Vertebrate Palaeoecology of the Pisco Formation (Miocene, Peru): Glimpses into the Ancient Humboldt Current Ecosystem

: The northward-ﬂowing Humboldt Current hosts perpetually high levels of productivity along the western coast of South America. Here, we aim to elucidate the deep-time history of this globally important ecosystem based on a detailed palaeoecological analysis of the exceptionally preserved middle–upper Miocene vertebrate assemblages of the Pisco Formation of the East Pisco Basin, southern Peru. We summarise observations on hundreds of fossil whales, dolphins, seals, seabirds, turtles, crocodiles, sharks, rays, and bony ﬁshes to reconstruct ecological relationships in the wake of the Middle Miocene Climatic Optimum, and the marked cooling that followed it. The lowermost, middle Miocene Pisco sequence (P0) and its vertebrate assemblage testify to a warm, semi-enclosed, near-shore palaeoenvironment. During the ﬁrst part of the Tortonian (P1), high productivity within a prominent upwelling system supported a diverse assemblage of mesopredators, at least some of which permanently resided in the Pisco embayment and used it as a nursery or breeding/calving area. Younger portions of the Pisco Formation (P2) reveal a more open setting, with wide-ranging species like rorquals increasingly dominating the vertebrate assemblage, but also local differences reﬂecting distance from the coast. Like today, these ancient precursors of the modern Humboldt Current Ecosystem were based on sardines, but notably differed from their present-day equivalent in being dominated by extremely large-bodied apex predators like Livyatan melvillei and Carcharocles megalodon .


Introduction
The coast of Peru is dominated by the Humboldt Current, which branches off the Antarctic Circumpolar Current and flows northwards along the Pacific margin of South America [1]. The Humboldt Current is thought to have originated during the Eocene or Oligocene, following major regional changes like the rise of the Andes and the opening of the Drake Passage that also contributed to Antarctic glaciation [2][3][4][5][6]. Today, its cold waters host an outstandingly productive upwelling system, supporting as much as one-fifth of the global marine fish catch [7][8][9].  [51] and Thornburg and Kulm [35]. (B) Schematic palaeogeographic map of the East Pisco Basin, showing the areas submerged during the Neogene (redrawn and modified from DeVries and Schrader [52]). (C) Schematic stratigraphic column of the Pisco Formation succession exposed in the East Pisco Basin, and its internal subdivision into allomembers (modified from Di Celma et al. [33]).

Stratigraphy of the Pisco Formation in the Lower Ica Valley
Previous studies have focused on exposures of the Pisco Formation along the lower Ica Valley, which features several hills (locally known as "cerros") with abundant fossil vertebrate assemblages [25,26]. The Pisco Formation in this area is about 470 m thick and comprises three depositional sequences/allomembers named P0, P1, and P2, respectively ( Figure 1C) [10,33,53,54]. Each allomember records a transgressive trend starting with a Glossifungites ichnofacies, coarse-grained lag deposits and shallow-water sandstones, which then give way to siltstones or diatomaceous mudstones reflecting suspension of organic-rich mud in a highly productive offshore shelf environment [10,33]. Volcanic ash layers, dolomitised mudstones, and thin, fine-to medium-grained sandstones are also locally present. Regressive facies successions are lacking [10]. The age of the P0 allomember has been poorly constrained until recently, but strontium isotope dating now places it in the middle Miocene, between 14.8 and 12.4 Ma [13]. Diatom biostratigraphy and 40 Ar/ 39 Ar ages date P1 to 9. 5-8.9 Ma and P2 to 8.5-6.7 Ma [10,14,47,[53][54][55]. Elsewhere, strata assigned to the Pisco Formation are thought to extend into the Pliocene [32,44].

Historical Rationale
Research on the fossil marine vertebrates from the Pisco Formation (or Pisco-equivalent strata) started in the Sacaco area with the Italian-born geographer and naturalist Antonio Raimondi, who emigrated to Peru in 1850 [56]. Later, Lisson [57] noted the presence of fossil whales in the lower Ica Valley, which a decade later was followed by the first description of the actual deposits and their lithology [58]. Colbert [59] named the first fossil vertebrate from the Pisco Formation, the basal delphinidan Incacetus broggii. Finally, Hoffstetter [60] published the first faunal assemblage (including cartilaginous and bony fishes, reptiles, birds, cetaceans, and seals) from the Sacaco area.
International interest in the Pisco Formation soared with the description of numerous marine vertebrates from the late 1970s onwards [15,16,20,46,48,50,. The richness and exceptional quality of these fossils in turn triggered attempts to quantify the overall association and investigate its taphonomy [17][18][19]31,34,88].

Field Methods
For some 15 field seasons, we conducted a detailed census of the vertebrate fossils at the localities of Cerro Colorado, Cerro Los Quesos, and Cerro Submarino [24][25][26], supplemented by data from Cerro Ballena, Cerro Blanco, Cerro Geoduck, Cerro Hueco la Zorra, Cerro La Bruja, Cerros Cadenas de los Zanjones, Cerros la Mama y la Hija, Corre Viento, Laguna Seca, and Quebrada del Toro Chico. We searched for fossils via systematic surface prospecting and, for each specimen, recorded: (i) its exact location, (ii) a preliminary identification, (iii) a concise description, and (iv) taphonomic observations regarding articulation, completeness, disposition and orientation, bite marks, associated faunal elements (including putative digestive tract content), the presence of a concretion, the extent of erosion, and associated sedimentary structures (see also Bosio et al. [30]); furthermore, whenever possible, we also recorded: (v) basic measurements (including the total length of the skeleton, and the condylobasal length and bizygomatic width of the cranium), and (vi) the degree of fusion of the vertebral epiphyses.
Identifications were often hindered by extensive weathering, especially on steep slopes and windswept flatlands. To ameliorate this problem, we partially excavated several specimens to expose further anatomical detail, and then covered them again with loose sediment to limit further erosion. Particularly informative fossils were collected and deposited in the Museo de Historia Natural de la Universidad Nacional Mayor de San Marcos (MUSM; Lima, Peru). Specimens accessioned or otherwise recorded at the MUSM prior to the beginning of this research effort were also included in our study.
To place our data into temporal context, we used a Jacob's staff and observations on strike and dip to create decimetre-scale sedimentary logs for 16 localities. We then summarised these logs into 15 composite stratigraphic columns ( Figure 2) and matched them with our fossil observations ( Figure 3). Specimens were placed along the sections with an accuracy ranging from ±0.4 to ±3.0 m. The database resulting from this combined taxonomic/taphonomic/temporal census is summarised below, and provided as a series of spreadsheets in Supplementary Table S1 (see also Bosio et al. [30]).

Figure 2.
Simplified geological map of the investigated exposures and localities of the Pisco Formation in the Ocucaje area (modified from Bosio et al. [30]). Locality names highlighted in bold indicate the 16 fossil-rich sites where thorough surveys for marine vertebrates were performed (see Supplementary Table S1).  [30]). Locality names highlighted in bold indicate the 16 fossil-rich sites where thorough surveys for marine vertebrates were performed (see Supplementary Table S1).
1 Figure 3. Stratigraphic sections (in metres) of 16 localities of the Pisco Formation in the Ocucaje area, showing the distribution of the prospected fossil vertebrates (modified from Bosio et al. [30]). The position of marker beds, intraformational unconformities, and radiometric ( 40 Ar/ 39 Ar) ages for dated volcanic ash layers are also indicated. Marker beds and dated ash layers after Di Celma et al. (in prep.) and Bosio et al. [14].

Palaeoecological Reconstruction of the Pisco Vertebrate Assemblage
The following Section 5.1 proposes a palaeoecological framework for the Miocene vertebrate assemblages from the EPB Pisco Formation. Sections 5.2 and 5.3 further elaborate on the lowest and highest levels of the associated food webs. Finally, Section 5.4 investigates the environmental causes behind the conservative taxonomic structure of the local elasmobranch faunas.

A Dynamic Palaeoecological and Palaeoenvironmental Landscape
Together, vertebrates, macroinvertebrates, and sedimentological data suggest a shallowwater (sub)tropical environment for the sandy strata of the middle Miocene P0 sequence [13,24]. One particularly tell-tale indicator is the extinct shark Hemipristis serra, which commonly occurs in Neogene low-latitude neritic deposits and whose closest extant relative-the snaggletooth shark Hemipristis elongata-inhabits tropical waters <30 m deep [104]. Hemipristis serra is relatively common in both P0 and the underlying Chilcatay Formation [105,106], but then vanishes from the local record. Elsewhere (including northern Peru [107]), this species persisted until the Pliocene at least. Its disappearance from the EPB may suggest a strengthening of the cold Humboldt Current, perhaps linked to major palaeoenvironmental changes that coincided with the deposition of P0: the onset of the Middle Miocene Climatic Transition and the emergence of the modern upwelling regime along the Pacific margin of South America [13,24].
Even though P0 is a coastal deposit, numerous fossils of large baleen whales and the occurrence of the common thresher shark (Alopias vulpinus) suggest strong offshore connections [24]. A rich assemblage of hard-shelled benthos [13], eagle rays and bony fish molariform teeth [24] evoke an ecosystem with abundant durophages and a welloxygenated sea bottom. Bite marks on both mysticetes and odontocetes indicate predation or scavenging by sharks [24,30], as also seen in the underlying Chilcatay Formation [105].
The depositional environment of P1, best known from Cerro Colorado, was also mostly shallow marine (though offshore deposition is witnessed by the diatomaceous upper part of the sequence) and home to a rich and ecologically complex vertebrate assemblage ( Figure 10) [90]. Microfossils, including diatoms, suggest prominent upwelling [108,109]. The cetacean assemblage is dominated by three species: the pontoporiid Brachydelphis mazeasi, the beaked whale Messapicetus gregarius, and an undescribed cetotheriid resembling (but distinctly larger than) Piscobalaena nana. Brachydelphis mazeasi was small, brevirostrine (sensu McCurry and Pyenson [110]), and likely a suction feeder [86]. If so, its prey would likely have consisted of fish, squid, and crustaceans small enough to be swallowed whole. Unlike its deep-diving extant relatives, M. gregarius has been interpreted as a raptorial predator targeting epipelagic schooling fishes like sardines (Sardinops cf. sagax [21]). Stomach contents show that the same applies to the unnamed cetotheriid [22,30]. The large number of specimens attributed to these three species identifies them as regular inhabitants of the late Miocene EPB, although they may have tracked seasonal prey distributions (see Section 5.2). In addition, the presence of extremely young individuals-a foetus of Brachydelphis, and calves of M. gregarius and the cetotheriid [83] (A.C., F.G.M., and G.Bi., pers. obs.)-suggest that the area may have served as a breeding/calving ground. Besides Brachydelphis, other delphinidans from P1 (Brujadelphis ankylorostris, Samaydelphis chacaltanae, and two putative kentriodontids) form a relatively minor but still relevant component of the marine mammal assemblage. They considerably vary in rostral proportions and total body length, from <2 m in S. chacaltanae to at least 3 m in the largest kentriodontid-like form, perhaps reflecting niche partitioning based on prey size [82]. Stem physeteroids (Acrophyseter sp., Livyatan melvillei) were considerably larger (from 4.0-4.3 m in Acrophyseter to 13.5 or even 16.2-17.5 m in Livyatan [80]) and likely apex predators alongside sharks such as Galeocerdo cuvier, Cosmopolitodus spp., and Carcharocles megalodon (see Section 5.3). Other similarly-sized cetaceans include at least two balaenopteroids, one of which had finely-fringed baleen consistent with foraging on extremely small prey like copepods [27]. In general, balaenopteroids are rare in the P1 sequence, perhaps reflecting a preference for more pelagic habitats.
The presence of two differently sized sulids (boobies) and a phalacrocoracid (shag/cormorant) evokes a near-shore environment and perhaps a degree of prey size-driven niche segregation between these fish eaters [75]. Several specimens of the sea turtle Pacifichelys urbinai indicate that at least some P1 tetrapods were durophagous [85]. Little is known about the crocodilian, but its mere presence is also consistent with a coastal setting.
Elasmobranch diversity varies throughout P1, with the older of the two main toothbearing intervals (ST-low1) being dominated by a rich community of warm-temperate mesopredators (especially copper sharks, Carcharhinus brachyurus) that used the shallow margins of the EPB as a secondary nursery ground [90,91]. The upper interval (ST-low2) preserves a simpler assemblage of presumably transient apex predators (Section 5.4) that may have exploited the deeper parts of the basin as a seasonal feeding ground. The nursery areas, which likely shifted spatially over time, were hotspots of diversity, disparity, and ecological complexity, and would have been characterised by relatively low predation pressure [90,91]. Ontogenetic shifts in diet and habitat preferences were likely crucial in promoting the persistence of such fragile elasmobranch communities within the EPB [91].
Bony fishes from P1 remain understudied, but include both pelagic (e.g., Sardinops and cf. Thunnus sp.) and coastal-neritic (e.g., Bagre, Cynoscion, and Sciaenidae indet.) forms [90]. Scales of Sardinops cf. sagax are widespread and sometimes preserved near or inside cetacean skeletons [21,22,30]. Sardines plausibly were a keystone species that helped to sustain the vertebrate food web of the Miocene EPB, and the main prey for a broad range of mysticetes, odontocetes, sea birds, sharks, and bony fishes (Section 5.2). The co-occurrence of ariids and sciaenids may indicate the former presence of a river mouth and/or swampy coastal woodlands in the vicinities of Cerro Colorado [90], as also suggested by numerous silicified wood fragments (some of which show Teredolites borings).
Within the lower part of the P2 sequence, the "CLB vertebrate level" evokes a marginal marine environment with abundant fish, bordered by a rocky shore hosting colonies of seals and penguins. The presence of large balaenopterids and billfishes implies strong offshore connections. Acrophyseter, Carcharocles, and Cosmopolitodus were likely the local top predators, and may have targeted smaller marine mammals and penguins on a seasonal basis (e.g., during the pupping season). At Cerro Colorado, for example, isolated teeth of Carcharocles and Cosmopolitodus often occur near pinniped and cetacean remains, perhaps reflecting predation or scavenging [26]. In addition, shark-bitten marine mammal skeletons are relatively frequent in the likely P2 sands exposed at Laguna Seca [30].
Above the lower P2 strata, fossil abundance again peaks in the diatomaceous package at the top of Cerro Los Quesos and roughly correlated horizons at localities such as Cerro Ballena, Cerro Blanco, and Cerro Hueco La Zorra. Seals are present but relatively uncommon. Balaenopterids belonging to at least three different species dominate the assemblage [10]. There are no obvious calves, contradicting earlier suggestions that these animals may have used the EPB as a breeding ground [111]. Cetotheriids are represented by Piscobalaena nana (also known from the Sacaco area [76]) and generally less common than balaenopterids. This is the inverse of the pattern seen in P1, and suggests even stronger links with the open ocean. The feeding strategy and diet of P. nana remain unclear, with potential prey species ranging from copepods to schooling fish [103].
Odontocetes are also comparatively rare, yet mostly different from those of the "CLB vertebrate level", indicating some degree of faunal turnover. Physeteroids include Acrophyseter sp., a second, larger macroraptorial species, and the first kogiids from the EPB: Platyscaphokogia landinii and Scaphokogia cochlearis [10,102]. While P. landinii was plausibly a raptorial feeder, the downwards-pointing, semi-cylindrical, pachyostotic rostrum of Scaphokogia suggests benthic foraging [102,112,113]. This interpretation is consistent with Scaphokogia being frequently encountered in shallow-water, near-shore deposits, both in the EPB and in the Sacaco area [114]. Beaked whales are rare, but include at least four species employing different feeding strategies: the suction-feeding crown ziphiid Nazcacetus urbinai, the raptorial stem ziphiid Chavinziphius maxillocristatus, and two unnamed species known only from fragmentary remains [79,84]. Pontoporiids and kentriodontids are absent (though Pliopontos occurs in seemingly younger strata of the Sacaco area [48]); they appear to have been locally replaced by small phocoenids (once again the oldest in the EPB), some of which resemble Lomacetus ginsburgi from the Sacaco area [15,66].
Elasmobranchs show little diversity and are mostly limited to large opportunistic apex predators like Carcharhinus leucas, Carcharocles megalodon, and Cosmopolitodus spp. [10,25]. Predation and/or scavenging of cetaceans by these sharks is attested by rare bite marks and the occasional occurrence of isolated teeth near mysticete skeletons [30].
Overall, palaeontological and sedimentological data suggest that the upper portion of P2 was deposited in a relatively distal setting characterised by high diatom productivity [108,109] and pelagic vertebrates with wide home ranges. These strata apparently reflect a deeper environment than is recorded in the underlying P1 sequence and the "CLB vertebrate level", and indeed likely are among the deepest deposits of the Pisco Formation west of the Ica River. Coastal taxa like seals, sulids and Scaphokogia are present but rare, suggesting that the top of P2 formed at the transition between the near-shore and pelagic realms. By contrast, the marginal-marine deposits of the Sacaco area have yielded terrestrial carnivorans such as procionids (Cyonasua sp. [115]), marine sloths (Thalassocnus spp. [70]), and greater numbers of Scaphokogia-like kogiids [112] and Piscobalaena nana [76]. As such, they may represent a more coastal equivalent of the upper P2 strata (see age estimates in Ochoa et al. [44]).

Was the Late Miocene EPB a Sardine-Based Microcosmos?
Three observations suggest that the late Miocene food web of the EPB was built on small epipelagic schooling fish ( Figure 10): first, exceptionally preserved stomach contents and regurgitations from a beaked whale, two cetotheriids, and a mackerel shark, all of which show that schooling sardines were a common prey item [21,22,30,112]; second, the regional occurrence of boobies [75], pelicans [116], and bronze sharks [90,91], all of which are known to target sardines and anchovies in present-day coastal upwelling settings [117][118][119]; and, finally, the widespread occurrence of loose cycloid scales resembling those of Sardinops sagax [22].
A similar ecological structure defines the modern Humboldt Current Ecosystem, in which abundant clupeiform fishes-the Peruvian anchoveta, Engraulis ringens, and the Pacific sardine, S. sagax-constitute a major food source [9,[120][121][122][123]. The same species also sustain the strategically important Peruvian fishery, which at times has produced up to 20% of the global fish catch from less than 1% of the total area of the global ocean [9]. The reasons behind these exceptional figures remain largely obscure, with primary productivity along the Peruvian coast being comparable to that of other major upwelling systems [9].
Previous explanations for the apparent decoupling between primary productivity and schooling fish abundance invoked short, efficient food chains, with small fish grazing directly on phytoplankton [7]. More recent data have shown, however, that both the Peruvian anchoveta and the Pacific sardine derive most of their dietary carbon from zooplankton [120,122]. Alternative reasons for high fish abundance may lie in: (i) a combination of high primary productivity and weak winds generating optimal growth conditions, (ii) a shallow oxygen minimum zone promoting zooplankton by concentrating its prey and excluding its predators, or (iii) El Niño-induced environmental variability favouring fast-growing organisms like diatoms, euphausiids, and clupeiform fishes, while at the same time inhibiting higher-level predators [9]. Similar mechanisms could plausibly have been at play during the late Miocene [124,125], when high-level predators were remarkably diverse and abundant [126], and thus explain the prominent role of schooling fish in the ancient EPB.
There are no modern analogues for the Miocene EPB along the Pacific coast of South America. Similarities do exist, however, with the Gulf of California: a semi-enclosed, internally subdivided basin with an important sardine fishery, located at the tropical end of an eastern boundary upwelling system [127][128][129][130]. Its resident sardines are spawned and attain sexual maturity in the deep waters of the central/southern gulf, but then move to the northern gulf to forage [127,128]. This may resemble the situation in the ancient EPB, where lateral advection of plankton [17,18] would have created an ideal feeding ground for adult sardines. Consistent with this idea, fossils of Sardinops cf. sagax from the Pisco Formation tend to be physically mature [21].

Carcharocles, Livyatan, and Other Overlords: What Ecological Role?
The structure of the late Miocene EPB ecosystem likely resembled that of its modern Humboldt Current descendant, but differed in at least one important regard: the presence of gigantic apex predators such as Livyatan melvillei and Carcharocles megalodon.
The ecology of "macroraptorial" sperm whales like Livyatan has been inferred from their functional anatomy, and is thought to have involved biting and tearing or shearing of large prey, rather than suction feeding [20,80,101,[131][132][133]. There is, however, as yet no direct evidence for this idea in the form of bite marks or fossilised stomach contents.
During the middle and late Miocene, macroraptorial sperm whales ranged in length from ca. 4.0 m in Acrophyseter to as much as 17.5 m in Livyatan [80], both of which occur in the Pisco Formation. There are no obvious modern analogues for these animals, with the only large mammal-eating cetacean of today-the killer whale Orcinus orca-being both smaller than the largest macroraptorial physeteroids and largely confined to higher latitudes [134]. Killer whales are also highly social and hunt cooperatively, a pattern that cannot be inferred (or discounted) for macroraptorial sperm whales, given the contrasting social habits of extant physeteroids [135,136]. Assuming solitary hunting, Acrophyseter was likely a grip-and-shear predator (sensu Peri et al. [126]) limited to relatively small prey like dolphins, seals, marine sloths, seabirds, and relatively large-sized fishes. By contrast, L. melvillei could plausibly have targeted larger species like cetotheriids by means of grip-and-tear feeding (sensu Berta and Lanzetti [137]), and likely overlapped in its diet with C. megalodon, a titanic otodontid shark whose maximum body length likely exceeded 15 m [138,139].
The feeding habits of C. megalodon have been widely discussed [140][141][142], also in the context of the late Miocene of Peru [114]. Despite its giant size, bite marks from the Sacaco area suggest that C. megalodon targeted relatively small marine mammals like the diminutive cetotheriid Piscobalaena nana and seals [114]. At first sight, this observation may seem at odds with teeth of C. megalodon showing lower calcium isotope values, and therefore signs of feeding at a higher trophic level, than the great white shark Carcharodon carcharias [143]. Yet these two findings are in fact consistent: most large marine mammals filter feed on tiny prey like copepods and krill, and thus occupy a low trophic level that results in relatively high calcium isotope ratios; by contrast, seals and small cetaceans tend to be dependent on longer food chains, which in turn are reflected in lower calcium isotope ratios. Overall, we hence suggest that C. megalodon was a mammal-eating predator (likely more predominantly so than C. carcharias) targeting small to medium-sized species, rather than the largest whales available as instead evoked elsewhere [140,144].
The coexistence of several megapredators like L. melvillei and C. megalodon may reflect ancient marine mammal size distributions. Baleen whales, in particular, were smaller during the Miocene than they are today, a pattern that only changed with the onset of the Northern Hemisphere glaciation and the extinction of most small species around 3 Ma [97,145,146]. Both C. megalodon and Livyatan-like physeteroids may have survived until roughly that time [147][148][149], when the decline of small mysticetes and various other marine vertebrates deprived them of their major food source [126,150,151].

A Refugium of Mackerel Sharks
Across the SE Pacific, Caribbean, and western Atlantic, Neogene ground sharks (Carcharhiniformes) tend to be more diverse than mackerel sharks (Lamniformes) [90,152]. This pattern may reflect the late Eocene spread of scleractinian coral reefs, which was paralleled by a similar radiation of ground sharks [153]. The P1 shark assemblage from the ST-low1 horizon of Cerro Colorado defies the broader trend by preserving more Lamniformes than Carcharhiniformes [90,91]. The reasons behind this phenomenon remain unclear, but may relate to the cold waters of the Humboldt Current preventing reef growth, as they likely do today [154,155], or to abundant marine mammals helping to sustain a flourishing lamniform fauna, as evoked by the occurrence of bite marks attributed to large mackerel sharks such as Cosmopolitodus on some cetacean skeletons [30]. Either way, the EPB (and perhaps other parts of the Pacific coast of South America) seemingly acted as a refugium, allowing the persistence of a conservative, lamniform-dominated shark fauna. The latter, in turn, likely gave rise to the great white shark, the largest macrophagous lamniform alive today [15,46,50,156].

Conclusions and Perspectives
Our palaeoecological reconstruction suggests that the Pisco Formation preserves conspicuous traces of a complex ecosystem driven by upwelling and abundant schooling fish.
As such, it may record an early phase of the modern Humboldt Current Ecosystem. The lower portions of the Pisco Formation (P0) formed in a warm, semi-enclosed, near-shore middle Miocene environment. During the first part of the early late Miocene (P1), high productivity driven by upwelling sustained a diverse assemblage of mesopredators, at least some of which permanently resided in the area and used it as a nursery or breeding/calving ground. Large megapredators like Livyatan melvillei and Carcharocles megalodon dominated the top of the food web. Niche partitioning and ontogenetic shifts in diet likely played key roles in maintaining a diverse and ecologically complex marine vertebrate community. Younger portions of the Pisco Formation (P2) reveal a more open setting bordering a rocky shore with seals and penguins. The marine vertebrate assemblage becomes increasingly dominated by wide-ranging species such as rorquals, but with local differences reflecting distance from the coast. Future research could help to elaborate these findings in several ways: (1) The P0 strata need to be investigated in greater detail to bridge the gap between the relatively well-known lower and upper Miocene assemblages of the EPB, and understand the impact of the Middle Miocene Climatic Optimum and Middle Miocene Climatic Transition. This will include prospecting additional sites such as Santa Rosa [59,96], the type locality of Incacetus broggi and Tiucetus rosae. (2) The rich shark assemblage from the ST-low1 interval of Cerro Colorado appears to have formed during a period of relative environmental stability, and as such provides an ideal opportunity to investigate feeding ecology and ontogenetic shifts in diet based on stable isotopes [143]. A similar approach could also be applied to toothed whales, e.g., with a view to corroborating the diet of extinct macroraptorial sperm whales and epipelagic beaked whales. (3) Despite its prominent ecological role, the deep-time history of the Pacific sardine remains largely obscure [157,158]. Abundant Sardinops-like fossils from the Neogene of Ecuador may help to rectify this, and elucidate the origin of the boosted fish productivity associated with the rise of the Humboldt Current Ecosystem. (4) Correlations between the EPB and the Sacaco area need to be improved to provide a clearer understanding of their respective ecological settings. At present, the radiometric dates from these two areas do not entirely match [96], with those from the lowermost strata of the Sacaco area being several decades old. (5) Comparisons of the Pisco Formation with coeval deposits elsewhere in the eastern Pacific and the Caribbean are currently sparse and ideally should focus on: (i) the Bahía Inglesa Formation of northern Chile (which shows obvious faunal similarities with both the EPB and the Sacaco area [159]), and (ii) the Miramar Formation and related deposits of northern Peru and Ecuador (which would elucidate the northern extent and impact of the ancient Humboldt Current). (6) Much systematic work remains to be done on the mysticetes, pinnipeds, and marine reptiles of the EPB, which ultimately will contribute to a more detailed understanding of the Pisco assemblages and their complex ecology. (7) Finally, a comprehensive assessment of the Palaeogene vertebrate assemblages of the EPB is still wanting. A broader knowledge of the fossil fishes and tetrapods of the Paracas and Otuma formations will elucidate the very roots of the present-day Humboldt Current Ecosystem.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jmse9111188/s1, Table S1. Dataset of fossil marine vertebrates from the Pisco Formation strata exposed at the study sites (precise GPS coordinates of the vertebrate finds are available on request from the authors).