Species identification of modern and archaeological shark and ray skeletal tissues using collagen peptide mass fingerprinting

Abstract

Introduction
Elasmobranchs, such as sharks and rays, are among the world’s most endangered vertebrates, with over 70% loss in abundance over the past 50 years due to human impacts. Zooarchaeological baselines of elasmobranch diversity, distribution, and exploitation hold great promise for contributing essential historical contexts in the assessment of contemporary patterns in their taxonomic diversity and vulnerability to human-caused extinction. Yet, the historical ecology of elasmobranchs receives relatively less archaeological attention compared to that of ray-finned fishes or marine mammals, largely due to issues of taxonomic resolution across zooarchaeological identifications.

Methods
We explore the use of Zooarchaeology by Mass Spectrometry (ZooMS) for species identification in this unstudied group, using an archaeological case study from the marine environments of the Florida Keys, a marine biodiversity hotspot that is home to an array of elasmobranch species and conservation efforts. By comparison with 39 modern reference species, we could distinguish 12 taxa within the zooarchaeological assemblage from the Clupper archaeological site (Upper Matecumbe Key) that included nine sharks, two rays and a sawfish.

Results and discussion
The results indicate that, through additional complexity of the collagen peptide mass fingerprint, obtained due to the presence of the cartilaginous type II collagen, ZooMS collagen peptide mass fingerprinting provides exceptionally high taxonomic resolution in this group, yielding species-level identifications in all cases where sufficient reference material was used. This case study also highlights the added value of ZooMS for taxa that are more difficult to distinguish in zooarchaeological analyses, such as vertebrae of the Atlantic sharpnose shark (Rhizoprionodon terraenovae) and the hammerhead sharks (Sphyrna spp.) in the Florida Keys. Therefore, the application of collagen peptide mass fingerprinting to elasmobranchs offers great potential to improve our understanding of their archaeological past and historical ecology.

Full text

Species identification of modern
and archaeological shark and ray
skeletal tissues using collagen
peptide mass fingerprinting
Michael Buckley
1
*, Ellie-May Oldfield
1
, Cristina Oliveira
2
,
Clara Boulanger
3,4
, Andrew C. Kitchener
5,6
, Nicole R. Fuller
2
,
Traci Ardren
7
, Victor D. Thompson
8
, Scott M. Fitzpatrick
9
and Michelle J. LeFebvre
2
1
School of Natural Sciences, Manchester Institute of Biotechnology, University of Manchester,
Manchester, United Kingdom,
2
Florida Museum of Natural History, University of Florida, Gainesville,
FL, United States,
3
Institute of Archaeology, University College London, London, United Kingdom,
4
UMR 7194
Histoire Naturelle de l’Homme Pre
´historique, De
´partement Homme et Environnement, Muse
´um National
d’Histoire Naturelle, Paris, France,
5
Department of Natural Sciences, National Museums Scotland,
Edinburgh, United Kingdom,
6
School of Geosciences, University of Edinburgh, Edinburgh, United Kingdom,
7
Department of Anthropology, University of Miami, Miami, FL, United States,
8
Georgia Museum of Natural
History and Department of Anthropology, University of Georgia, Athens, GA, United States,
9
Department of
Anthropology, University of Oregon, Eugene, OR, United States
Introduction: Elasmobranchs, such as sharks and rays, are among the world’s
most endangered vertebrates, with over 70% loss in abundance over the past 50
years due to human impacts. Zooarchaeological baselines of elasmobranch
diversity, distribution, and exploitation hold great promise for contributing
essential historical contexts in the assessment of contemporary patterns in
their taxonomic diversity and vulnerability to human-caused extinction. Yet,
the historical ecology of elasmobranchs receives relatively less archaeological
attention compared to that of ray-finned fishes or marine mammals, largely due
to issues of taxonomic resolution across zooarchaeological identifications.
Methods: We explore the use of Zooarchaeology by Mass Spectrometry (ZooMS)
for species identification in this unstudied group, using an archaeological case
study from the marine environments of the Florida Keys, a marine biodiversity
hotspot that is home to an array of elasmobranch species and conservation
efforts. By comparison with 39 modern reference species, we could distinguish
12 taxa within the zooarchaeological assemblage from the Clupper
archaeological site (Upper Matecumbe Key) that included nine sharks, two rays
and a sawfish.
Results and discussion: The results indicate that, through additional complexity
of the collagen peptide mass fingerprint, obtained due to the presence of the
cartilaginous type II collagen, ZooMS collagen peptide mass fingerprinting
provides exceptionally high taxonomic resolution in this group, yielding
species-level identifications in all cases where sufficient reference material was
Frontiers in Marine Science frontiersin.org01
OPEN ACCESS
EDITED BY
Anna Rita Rossi,
Sapienza University of Rome, Italy
REVIEWED BY
Alberto Sa
´nchez-Gonza
´lez,
National Polytechnic Institute (IPN), Mexico
Thomas J. Kean,
University of Central Florida, United States
*CORRESPONDENCE
Michael Buckley
m.buckley@manchester.ac.uk
RECEIVED 23 September 2024
ACCEPTED 24 October 2024
PUBLISHED 26 November 2024
CITATION
Buckley M, Oldfield E-M, Oliveira C,
Boulanger C, Kitchener AC, Fuller NR,
Ardren T, Thompson VD, Fitzpatrick SM and
LeFebvre MJ (2024) Species identification
of modern and archaeological shark
and ray skeletal tissues using collagen
peptide mass fingerprinting.
Front. Mar. Sci. 11:1500595.
doi: 10.3389/fmars.2024.1500595
COPYRIGHT
©2024Buckley,Oldfield, Oliveira, Boulanger,
Kitchener, Fuller, Ardren, Thompson, Fitzpatrick
and LeFebvre. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 26 November 2024
DOI 10.3389/fmars.2024.1500595

used. This case study also highlights the added value of ZooMS for taxa that are
more difficult to distinguish in zooarchaeological analyses, such as vertebrae of
the Atlantic sharpnose shark (Rhizoprionodon terraenovae) and the hammerhead
sharks (Sphyrna spp.) in the Florida Keys. Therefore, the application of collagen
peptide mass fingerprinting to elasmobranchs offers great potential to improve
our understanding of their archaeological past and historical ecology.
KEYWORDS
collagens, cartilage, ZooMS, species identification, Florida Keys, proteomics
Introduction
Elasmobranchs (sharks, skates, and rays) are essential to the
health of marine ecosystems, with many (particularly sharks) often
playing vital roles as keystone taxa and apex predators, helping to
maintain species diversity and ecosystem health (Heithaus et al.,
2007;Motivarash Yagnesh et al., 2020). Today, despite having existed
for over 400 million years, elasmobranchs are among the world’s
most endangered vertebrate groups due to human impacts, with over
70% loss in abundance over the past 50 years (Pacoureau et al., 2021).
This has been particularly devastating to shark populations, whose
life histories are characterized by low fecundity, slow growth, late
maturity, and relatively long-life spans, all of which makes them
particularly vulnerable to population decline via anthropogenic
threats such as over-fishing, habitat degradation, and accelerated
climate change (Dulvy et al., 2021;Field et al., 2009;Sherman et al.,
2023). Moreover, increases in shark exploitation are also linked to
declines in popular bony fishes available for harvest (Camhi et al.,
1998), as well as rising demand within the international shark fin
trade network (Worm et al., 2024).
The historical ecology of elasmobranchs, and especially sharks,
demonstrates that humans have engaged with and harvested taxa
across the world for millennia (e.g., the Mediterranean [Giovos
et al., 2021;Mojetta et al., 2018]; the North Sea [Bom et al., 2022];
the Gulf of Mexico [Martı

nez-Candelas et al., 2020]; Southeast Asia
[Boulanger, 2023;Boulanger et al., 2021]). Utilizing historical
perspectives of shark diversity and harvest patterns, garnered
through sources such as oral histories (including traditional
ecological knowledge, and indigenous traditional ecological
knowledge), ethnohistoric texts, art, photography, and more
recent (e.g., decade-scale) survey data, make it clear that the
worldwide decline in taxa has been driven by human overfishing,
with particular emphasis on loss over the past century (e.g.,
Bradshaw et al., 2008;Juan-Jordaet al., 2022;Roff et al., 2018).
This relatively recent timescale for diversity loss presents challenges
for creating long-term baselines needed in conservation decision-
making and human-wildlife conflict management, whereby
historical patterns of species diversity, distribution (including
habitat use), and community composition for many taxa are
either understudied or unknown (e.g., Ferretti et al., 2008,2018;
Heithaus et al., 2007;McClenachan et al., 2016). This is particularly
the case for century-to-millennia-scales of comparison (e.g., Burg
Mayer & de Freitas, 2023).
Archaeology has much to contribute to deep-time baselines and
elucidating the antiquity of human engagements with elasmobranch
taxa through the study of zooarchaeological specimens. Robust
zooarchaeological shark, and other elasmobranch, assemblages have
been recovered from many archaeological sites across the world (the
Americas [e.g., Prieto, 2023;Betts et al., 2012;Colvin, 2014;Rick
et al., 2002;Gilson and Lessa, 2021;Lopes et al., 2016], the African
continent and Arabian peninsula [e.g., Charpentier et al., 2020;
Roberts et al., 2019], the Mediterranean [van Neer et al., 2005]
across Oceania [Wright et al., 2016;Weisler and McNiven, 2016]
and Asia [e.g., Boulanger, 2023;Langley et al., 2023;Boulanger et al.,
2023,2022,2021,2019;Kealy et al., 2020;O’Connor et al., 2019]).
However, relative to bony fish specimens and other types of marine
vertebrate taxa, zooarchaeological elasmobranch specimens are
often difficult to identify beyond order, family, or genus based on
morphology alone (Ono and Intoh, 2011), making it challenging to
construct deep-time baselines with enough taxonomic resolution to
support conservation needs. There are several reasons for this, but
primarily it is because chondrichthyan skeletons are composed of
hyaline cartilage, with some elements only partly calcified. As a
result, it is the vertebral centra, teeth, spines, and dermal denticles
that are mostly preserved in archaeological deposits, and although
these are the most readily distinguishable elasmobranch parts, they
are particularly difficult to identify to the species level due to
uniformity and ubiquity among elements within and between
many species (e.g., Rick et al., 2002). For example, centrum size,
shape and number of vertebrae can be variable among individuals of
the same species as well as between species (Springer & Garrick,
1964). Thus, ease of identification to species and quantifications of
relative abundance (e.g., % number of identified specimens [NISP],
% minimum number of individuals [MNI]) may not be
straightforward between regions of the same vertebral column.
Shark tooth morphology also differs interspecifically and
depending on tooth position along the upper and lower jaws
within species. Furthermore, it is important to note that
Buckley et al. 10.3389/fmars.2024.1500595
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challenges of specimen identification may also be compounded by
lack of access to adequate (e.g., taxonomically and elementally
representative) modern comparative skeletal collections for many
shark species (Burg Mayer and de Freitas, 2023;Shepherd and
Campbell, 2021). All these factors mean that it can be difficult to
identify elasmobranch zooarchaeological specimens, and even more
so within assemblages from world regions characterized by high
elasmobranch taxonomic diversity within orders, families, and
genera (e.g., Carcharhinus spp., Sphyrna spp.).
Motivated by a desire to better leverage zooarchaeological data
within elasmobranch archaeology and historical ecological fisheries
baselines, we present the results of an initial Zooarchaeology by
Mass Spectrometry (ZooMS) analysis of elasmobranch specimens
from a zooarchaeological assemblage at the Clupper site (8MO17).
Clupper is an Ancestral Period
1
indigenous archaeological site
located in the Florida Keys (Keys), USA (Figure 1); the Keys
comprise a small-island archipelago renowned for its breadth of
marine biodiversity, including several elasmobranch species
(Table 1). The marine and estuary habitats of the Keys are
essentially made up of “Bay”and “Ocean”waters, including
shallower waters (e.g., < 2 m deep) protected by mangrove islands
and deeper waters (e.g., 10-30 m deep) beyond embayments,
respectively (Tinari and Hammerschlag, 2021).
The Keys can be characterized as a historical fishery with
shifting baselines of fisheries health and persistent decline across a
diversity of species throughout the 20
th
century (e.g., Ault et al.,
2005;McClenachan, 2009), including sharks, rays, and sawfishes
(Heithaus et al., 2007;Powers et al., 2013). Zooarchaeological
assemblages from the Keys provide a record of deep-time
fisheries, including centennial-to millennial-scale perspectives of
taxonomic diversity, distribution, and human engagement (e.g.,
LeFebvre et al., 2022), but given the abovementioned limitations in
the zooarchaeology of elasmobranch remains, the goals of this study
were to: 1) investigate the efficacy of the collagen-fingerprint-based
approach known as ZooMS (Zooarchaeology by Mass
Spectrometry) in identifying zooarchaeological elasmobranch
specimens to species; and 2) begin to build a species-level baseline
of elasmobranch diversity present in the Keys approximately 700-
1,000 years ago (Ardren et al., 2018) for the advancement of
archaeological and historical ecological research.
We begin with background summaries of biomolecular
methods in species identifications as well as elasmobranch skeletal
structure to contextualize the ZooMS approach, followed by an
overview of the Clupper site and current zooarchaeological analysis
of elasmobranch specimens. Then we review the materials and
methods of analysis with an emphasis on building a modern
comparative baseline for collagen peptide mass fingerprints of
this previously untried taxonomic group. The results of the
analysis demonstrate the ability of ZooMS to achieve species-level
identifications of zooarchaeological specimens and are presented
within the context of contemporary elasmobranch diversity and
habitats. Using the Clupper data as an exemplar, we highlight the
implications of the results from archaeological perspectives of
Ancestral Indigenous harvest, elasmobranch historical ecology,
and future zooarchaeological research.
Background
Biomolecular methods of
species identification
Although biomolecular methods offer an objective solution to
the issue of species identification, they are largely underutilized for
several reasons, including costs per sample, damage to specimens,
and speed of interpretation in some cases. The analysis of ancient
DNA is becoming increasingly more cost-effective (e.g., Seersholm
et al., 2018), and will undoubtedly increase in use in the future, yet
its greatest limitation is that of molecular preservation, whereby the
few studies that have attempted to do so for shark taxa have focused
on very recent material less than a few hundred years old (Ahonen
and Stow, 2008;Nielsen et al., 2017) and even these can yield <50%
success rates (e.g., Shepherd and Campbell, 2021).
Proteins on the other hand, particularly bone collagen, are
thought to survive much longer, or at least better in warmer
environments, such as those inhabited by most tropical shark
species. Harnessing this greater longevity, a method of species
identification by ‘fingerprinting’collagen using mass
spectrometry, referred to as ‘ZooMS’(Zooarchaeology by Mass
Spectrometry), was created over a decade and a half ago (Buckley
et al., 2009;2010), based initially on mammals for investigating
animal husbandry practices (e.g., Buckley and Kansa, 2011;Price
et al., 2013), with wider taxonomic use in studies on reptiles
(Harvey et al., 2019;Guiry et al., 2024), birds (Eda et al., 2020),
amphibians (Buckley and Cheylan, 2020), and fishes (Richter et al.,
2011;Harvey et al., 2018;2022;Rick et al., 2019;Guiry et al., 2020;
Buckley et al., 2021;2022) in comparison to the other ‘lower
vertebrate’groups.
Although there are more than 28 types of collagen known to
exist within humans at some point in life (Ricard-Blum, 2011), 80-
90% of the collagen in the body consists of types I, II and III, of
which type I collagen is by far the most abundant. The triple helical
structure common to all collagens is maintained through the
presence of repeating amino acids, proline (Pro) and its modified
form hydroxyproline (Hyp), which induce the twisting structure in
each chain. Some collagen types are formed from three identical
trains, with the cartilage-dominant type II collagen being a prime
example. In contrast, the dominant type in bone, type 1 collagen, is
made of genetically distinct chains, which in most vertebrates are
made up of two identical chains (alpha 1 chains) and one genetically
distinct (alpha 2) chain. In bony ray-finned fishes, type 1 collagen is
composed of three distinct chains, where the third (a3(I)) is a
duplicate of the (a1(I)) gene (Morvan-Dubois et al., 2003).
However, this distinct third chain does not appear to exist in
1 Here, within Florida archaeology, the Ancestral Period refers to time spans
prior to European colonization. The use of this term as a chronological
descriptor of Indigenous history is supported and preferred by some U.S.
federally recognized contemporary Indigenous Peoples, including the
Seminole Tribe of Florida in south Florida (see https://stofthpo.com/
seminole-history/).
Buckley et al. 10.3389/fmars.2024.1500595
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sharks and lampreys (Kimura and Ohno, 1987), given the timing of
the duplication event that gave rise to this (Harvey et al., 2021).
Elasmobranchs and their skeletal structure
The vertebrates are a subphylum (Vertebrata) within the phylum
Chordata. The jawed vertebrates (gnathostomes) are a group
comprising all tetrapods as well as the bony fishes (Osteichthyes),
and the cartilaginous fishes (Chondrichthyes). During ontogeny,
most vertebrate skeletons are initially composed mostly of hyaline
cartilage that is largely replaced by bone via endochondral
ossification (Hall, 1975); the biomineralization of skeletal tissues
occurs through the deposition of biological apatite into collagen-rich
(or amelogenin-rich, in the case of enamel and enameloid) matrices
(Donoghue et al., 2006). However, as their name suggests, the
skeletal structure of cartilaginous fishes remains primarily
composed of cartilage, as they do not develop osseous skeletons,
having secondarily lost this ability to produce endoskeletal bone
(Coates et al., 2008). Elasmobranchs develop a relatively thin outer
layer of cortical mineralization over most of their skeleton (Dean
et al., 2015;Seidel et al., 2016), which is typically characterized by
type II collagen, a triple helical molecule made up of three identical
alpha chains (COL2A1).
The living chondrichthyans are split into two main groups, the
Holocephali and the Elasmobranchii. The former are divided into
three families, the Callorhincidae, Chimaeridae and the
Rhinochimaeridae, whereas the latter are a much more diverse
group, including the sharks (selachians; nine recognized orders)
and the rays, skates, sawfishes and guitarfishes (batoids; four
recognized orders). The cartilaginous elements (jaws, fins, and
vertebrae) are covered by mineralized polygonal tiles called
tesserae, more abundant in elasmobranchs, but also increasingly
recognized in holocephalans (Pears et al., 2020;Seidel et al., 2020). It
is the internal part of these tesserae, the round cells enclosed in
lacunae, that contain the cartilaginous type II collagen (Seidel et al.,
2017), whereas the external parts located on the perichondrial side
are characterized by flatter cells that are engulfed in a type I collagen
matrix (Orvig, 1951;Kemp and Westrin, 1979), known as fibrous
perichondrium (Dean et al., 2015). In addition to this, as reviewed
by Dean and Summers (2006), there are other types of mineralized
tissues in the elasmobranch endoskeleton. Areolar mineralization
characterizes the vertebral centra, and lamellar mineralization,
comparable to bone tissue, has been recorded in the neural
arches. The latter was first termed osseous tissue due to the
presence of elongated cells like the osteoblasts of bone (Peignoux-
Deville et al., 1982), expressing type I collagen genes (Enault et al.,
2016), and because they are enclosed within a type I collagen-rich
extracellular matrix that is able to mineralize (Eames et al., 2007);
see Berio et al. (2021) for review. Therefore, although dominated by
type II collagen, type I collagen typical of bone is estimated to
account for about one third of the total collagen content of shark
cartilage (Rama & Chandrakasan, 1984).
The Clupper site and
elasmobranch specimens
Elasmobranch specimens and artifacts, especially from sharks
(e.g., shark tooth drills), have been documented from coastal, island,
FIGURE 1
Location of the Clupper archaeological site (8MO17) within the Upper Keys, and distinctions between Upper, Middle, and Lower Keys.
Buckley et al. 10.3389/fmars.2024.1500595
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and inland Ancestral Period Indigenous archaeological sites across
greater Florida, attesting to the cultural significance of such taxa in
the past, including their use as food, tools, and items of personal
adornment, ritual significance, and/or trade for several millennia
(e.g., Early Archaic, ca. 6,000 BC [Farrell, 2021]; see also Keller and
Thompson, 2013). The zooarchaeological specimens used in this
study were selected from the Clupper site, an Indigenous village
inhabited ca. AD 650-1,250 (Ardren et al., 2018)(Figure 1). The site is
located on Upper Matecumbe Key, surrounded by or within
proximity to a variety of maritime habitats, including sandy bottom
flats, coral reefs, sea grass beds, rocky substrate, mangroves, marshes,
and deeper waters. The site comprises a black earth midden. The
midden is characterized by an abundance of well-preserved vertebrate
fauna, invertebrate fauna, pottery and shell artifacts (Ardren et al.,
2018;LeFebvre et al., 2022). Current zooarchaeological results from
midden samples show that while bony fishes are the most abundant
vertebrate taxon as represented per number of individual specimens
(NISP = 9,350), followed by marine and freshwater turtles (NISP =
7,683), elasmobrachs are the third most relatively well-represented
taxon with 578 NISP (Oliveira, 2024;seeSupplementary Table S1).
However, it is possible that elasmobranchs are underrepresented
within the assemblage due to their cartilaginous structure, with
representation being limited to vertebrae and teeth (LeFebvre et al.,
2022;Rick et al., 2002).
The Clupper elasmobranch specimens were identified based on
morphology using modern comparative skeletal specimens from the
Environmental Archaeology Program (EAP) and South Florida
Archaeology collections at the Florida Museum of Natural
History, USA (LeFebvre et al., 2022). The taxonomic resolution
achieved (Table 2) included specimens identified at the level of
order (n = 2), family (n = 1), genus (n=3), species (n=4), and cf.
(compares with) species (n=3). Order-level identifications to
Carcharhiniformes (ground sharks; n = 229) and Rajiformes
(flattened cartilaginous fishes; n = 101) accounted for the
majority of individual specimen identifications. Carcharhinidae
was the only family-level identification. The most abundantly
identified shark taxon beyond order or family was the
hammerhead genus (Sphyrna spp.). Spotted eagle ray (Aetobatus
narinari) specimens were the most common Rajiformes identified
beyond order. Other levels of genus, species, and cf. species
identifications included nurse shark (Ginglymostoma cirratum),
tiger shark (Galeocerdo cuvier), sandbar shark (cf. Carcharhinus
plumbeus), lemon shark (cf. Negaprion brevirostris), Atlantic
sharpnose shark (Rhizoprionodon terraenovae), sawfish (Pristis
TABLE 1 List of shark and ray species observed through contemporary survey efforts in south Florida (e.g., Miami and Florida Keys).
Family Species Common Name Conservation status
Ginglymostomatidae Ginglymostoma cirratum
+
nurse shark Vulnerable
Lamnidae Carcharodon carcharias white shark Vulnerable
Carcharhinidae Carcharhinus acronotus blacknose shark Endangered
Carcharhinidae Carcharhinus brevipinna spinner shark Vulnerable
Carcharhinidae Carcharhinus leucas bull shark Vulnerable
Carcharhinidae Carcharhinus limbatus
+
blacktip shark Vulnerable
Carcharhinidae Carcharhinus obscurus
+
dusky shark Endangered
Carcharhinidae Carcharhinus perezi Caribbean reef shark Endangered
Carcharhinidae Carcharhinus plumbeus sandbar shark Endangered
Carcharhinidae Negaprion brevirostris
+
lemon shark Vulnerable
Carcharhinidae Rhizoprionodon terraenovae
+
Atlantic sharpnose shark Least concern*
Galeocerdonidae Galeocerdo cuvier
+
tiger shark Near threatened
Sphyrnidae Sphyrna lewini
+
scalloped hammerhead Critically endangered
Sphyrnidae Sphyrna mokarran great hammerhead Critically endangered
Sphyrnidae Sphyrna tiburo
+
bonnethead shark Endangered
Pristidae Pristis pectinata
+
smalltooth sawfish Critically endangered
Dasyatidae Hypanus americanus southern stingray Near Threatened
Urotrygonidae Urobatis jamaicensis yellow round ray Least concern*
Aetobatidae Aetobatus narinari
+
whitespotted eagle ray Endangered
Data compiled from Heithaus et al. (2007);Tinari and Hammerschlag (2021), and Ramey (2021). Conservation status from IUCN (May 16, 2024). *IUCN population trends for all taxa are listed
as “decreasing”except for Atlantic sharpnose shark as “increasing”and yellow round ray as “stable.”
+
Species identified by ZooMS in the Clupper zooarchaeological elasmobranch assemblage.
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spp.), stingray (Hypanus spp.), and Atlantic stingray (Hypanus
cf. sabinus).
Interpretations of Indigenous fishing practices at Clupper
indicate emphases on inshore habitats and a generalist, or multi-
species approach, to fishing versus targeted or selective (LeFebvre
et al., 2022;Oliveira, 2024). However, it is important to note that a
generalist (i.e., multi-species) fishing strategy does not preclude
targeted or preferential consumption of species caught. Rather, it
refers to fisheries strategies that usually employ gear suited to
capturing a diversity of species versus single or a selected few
(Bieg et al., 2018). It is also the case that generalist fishing strategies
are often not exclusively practiced and may include selective fishing
approaches depending on species behaviors and availability (e.g.,
migrations, aggregations), as well as environmental conditions
throughout a year (e.g., Ziegler et al., 2018).
The suggestion of a generalist strategy to fishing at Clupper is
based on the identifications of bony fish and sea turtle taxa at genus
or species levels and the overall high taxonomic diversity present in
the zooarchaeological assemblage (LeFebvre et al., 2022). Moreover,
genus- and species-level identifications indicate a fishery primarily
focused on inshore habitats (Oliveira, 2024). Patterns of harvest
over time have also been noted, including a decrease in almost
exclusively marine taxa (e.g., sea turtles, grunts [Haemulon spp.])
concomitant with an increase in taxa that also frequent brackish
waters, such as catfishes (e.g., Ariopsis felis and Bagre marinus)
(Oliveira, 2024). Based on the schooling habits and daily migration
patterns of the majority of bony fishes identified, fishing technology
likely employed a mix of mass capture with nets and smaller catches
via traps (Oliveira, 2024). Interpreting elasmobranch harvest has
been less nuanced due to the lack of taxonomic resolution beyond
order or family. Thus, shark, ray, and sawfish species harvest has
been generally described as possible across essentially any habitat a
shark or ray may inhabit (LeFebvre et al., 2022).
Materials and methods
Both modern and zooarchaeological elasmobranch specimens
were sampled for this study. The selection and analysis of modern
specimens was based on establishing a modern baseline of species
identification, using ZooMS. Zooarchaeological specimens were
selected to test the ability of ZooMS to confirm, refine, or refute
previous identifications based on morphology.
Modern reference materials
Modern skeletal tissues (jaw and/or vertebrae; see
Supplementary Table S2) were obtained for 39 species of 17
taxonomic families of elasmobranch, including all of those known
to inhabit the oceanic and estuarine environments surrounding
peninsular Florida and the Keys (Table 3). The specimens were
selected from the modern skeletal comparative collection in the
Environmental Archaeology Program at the Florida Museum of
Natural History supplemented by specimens of two Pristis species
sampled in triplicate from National Museums Scotland due to
uncertainty in the taxonomic identification of the Florida
Museum of Natural History (FLMNH) reference specimen. With
a focus on sampling as much taxonomic diversity as possible
relevant to the study region represented in the comparative
collection, specimens included vertebrae, teeth, as well as dry-
preserved cartilaginous materials.
Archaeological materials
Selection of zooarchaeological specimens from currently
analyzed Clupper samples was aimed at either refining or testing
the taxonomic breadth of specimen identifications made based on
morphology (Supplementary Table S3). Specimens were analyzed
from the following levels of identification: Order
(Carcharhiniformes, Rajiformes), Family (Carcharhinidae), Genus
(Sphyrna sp., Hypanus sp., Pristis sp.), species (Aetobatus narinari,
Rhizoprionodon terraenovae,Ginglymostoma cirratum), and likely
species (cf. Hypanus sabinus,cf.Negaprion brevirostris,cf.
Galeocerdo cuvier). Analyzed specimens were from two 50 × 50
cm test pits excavated in 10 cm arbitrary levels. It is important to
note that the focus of this initial effort was not quantitative in terms
of identifying relative abundances of species represented at the site
or trends in elasmobranch taxonomic diversity through time (i.e.,
across levels of excavation), but rather to gain as refined a
taxonomicbaselineaspossibleofspeciesrepresentedatthe
Clupper site during its Ancestral history from which to (re)
consider continuing and future approaches to elasmobranch
identification, quantification, and interpretation at Clupper and
across the Keys more broadly.
TABLE 2 Elasmobranch taxonomic identifications based on morphology
from the Clupper site, Upper Matecumbe Key, Florida, U.S.A. Levels of
taxonomic identification span order to species.
Taxonomic ID Level
of ID
Common Name NISP
Ginglymostoma cirratum species Nurse shark 25
Carcharhiniformes order Ground sharks 229
Carcharhinidae family Requiem sharks 40
cf. Carcharhinus plumbeus cf. species Sandbar shark 1
cf. Negaprion brevirostris cf. species Lemon shark 4
Rhizoprionodon terraenovae species Atlantic sharpnose shark 10
Galeocerdo cuvier species Tiger shark 2
Sphyrna sp. genus Hammerhead shark 124
Pristis sp. genus Sawfish 16
Rajiformes order flattened cartilaginous fish 101
Hypanus sp. genus Stingray 4
Hypanus cf. sabinus cf. species Atlantic stingray 7
Aetobatus narinari species Spotted eagle ray 15
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TABLE 3 List of species included in this study (specimen details given in Supplementary Table S2).
Order Family Common Name Scientific Name
Hexanchiformes Hexanchidae Bluntnose sixgill shark Hexanchus griseus
Orectolobiformes Ginglymostomatidae Nurse shark Ginglymostoma cirratum*
Lamniformes Carchariidae Sand tiger shark Carcharias taurus
Lamniformes Lamnidae Great white shark Carcharodon carcharias*
Lamniformes Lamnidae Shortfin mako shark Isurus oxyrinchus
Lamniformes Alopiidae Common thresher shark Alopias vulpinus
Carcharhiniformes Triakidae School shark/Tope shark Galeorhinus galeus*
Carcharhiniformes Triakidae Dusky smooth-hound shark Mustelus canis
Carcharhiniformes Hemigaleidae Snaggletooth shark Hemipristis elongata*
Carcharhiniformes Carcharhinidae Blacknose shark Carcharhinus acronotus
Carcharhiniformes Carcharhinidae Bignose shark Carcharhinus altimus
Carcharhiniformes Carcharhinidae Spinner shark Carcharhinus brevipinna
Carcharhiniformes Carcharhinidae Galapagos shark Carcharhinus galapagensis
Carcharhiniformes Carcharhinidae Finetooth shark Carcharhinus isodon
Carcharhiniformes Carcharhinidae Bull shark Carcharhinus leucas
Carcharhiniformes Carcharhinidae Blacktip shark Carcharhinus limbatus*
Carcharhiniformes Carcharhinidae Dusky shark Carcharhinus obscurus
Carcharhiniformes Carcharhinidae Caribbean reef shark Carcharhinus perezii
Carcharhiniformes Carcharhinidae Sandbar shark Carcharhinus plumbeus
Carcharhiniformes Carcharhinidae Night shark Carcharhinus signatus
Carcharhiniformes Carcharhinidae Lemon shark Negaprion brevirostris*
Carcharhiniformes Carcharhinidae Blue shark Prionace glauca*
Carcharhiniformes Carcharhinidae Atlantic sharpnose shark Rhizoprionodon terraenovae
Carcharhiniformes Galeocerdonidae Tiger shark Galeocerdo cuvier*
Carcharhiniformes Sphyrnidae Scalloped hammerhead Sphyrna lewini
Carcharhiniformes Sphyrnidae Great hammerhead Sphyrna mokarran
Carcharhiniformes Sphyrnidae Bonnethead Sphyrna tiburo
Carcharhiniformes Sphyrnidae Smooth hammerhead Sphyrna zygaena
Rhinopristiformes Rhinobatidae Atlantic guitarfish Pseudobatos lentiginosus
Rhinopristiformes Pristidae Largetooth sawfish Pristis pristis
Rhinopristiformes Pristidae Smalltooth sawfish Pristis pectinata
Rajiformes Rajidae Clearnose skate Rostroraja eglanteria*
Myliobatiformes Dasyatidae Roughtail stingray Bathytoshia centroura
Myliobatiformes Dasyatidae Southern stingray Hypanus americanus
Myliobatiformes Dasyatidae Atlantic stingray Hypanus sabinus*
Myliobatiformes Dasyatidae Bluntnose stingray Hypanus say
Myliobatiformes Urotrygonidae Yellow stingray Urobatis jamaicensis
Myliobatiformes Aetobatidae Spotted eagle ray Aetobatus narinari
Myliobatiformes Rhinopteridae Cownose ray Rhinoptera bonasus
*Specimens studied for LC-MS/MS sequencing to improve marker identification.
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ZooMS collagen peptide
mass fingerprinting
Approximately 25-50 mg of modern and archaeological tissues
were processed for collagen peptide mass fingerprinting following
Buckley (2013), with modern tissues degreased beforehand by fully
submerging twice in 83%/17% chloroform/methanol (first for 20
minutes, then for 3 hours). For collagen extraction, 1 mL 0.6 M
hydrochloric acid (Sigma-Aldrich, UK) was added to each intact
sample to enable decalcification. Half of the acid-soluble fraction
was then ultrafiltered into 50 mM ammonium bicarbonate (Sigma-
Aldrich, UK), with two centrifugation steps (20 min; 12,400 rpm)
and recovered in 0.1 mL solution. This was then diluted 1/20 and 1
µL co-crystalized with 1 µL 10 mg/mL alpha-cyano
hydroxycinnamic acid (Sigma-Aldrich, UK) in 50% acetonitrile
(ACN)/0.1% trifluoroacetic acid (TFA) onto a stainless-steel
Matrix Assisted Laser Desorption Ionization Time-of-Flight
(MALDI-ToF) mass spectrometric target plate. Using a Bruker
Rapiflex MALDI-ToF instrument, up to 20,000 laser shots were
acquired over the mass/charge (m/z) range 700-3,700 and resultant
spectra of archaeological samples were manually categorized into
‘groups’that each were composed of their own set of peptide
markers. Peaks that appeared to differ between these groups were
then compared with those present or absent in the associated
reference material within this study, taking into consideration
morphological identification for the majority in each
archaeological group. Tandem mass spectra were also acquired
via LC-MS/MS sequencing of selected modern specimens for
improved results from database searching to assist confirmation
of homologous markers between taxa.
LC-MS/MS sequencing
LC-MS/MS was carried out to improve understanding of the
peaks observed in the ZooMS spectra (see Supplementary Table S3
for taxa selected to span range of biomarkers). Digested samples
were analyzed using an UltiMate®3000 Rapid Separation LC
(RSLC, Dionex Corporation, Sunnyvale, CA) coupled to a QE HF
(Thermo Fisher Scientific, Waltham, MA) mass spectrometer.
Mobile phase A was 0.1% formic acid in water and mobile phase
B was 0.1% formic acid in acetonitrile, and the column used was a
75 mm × 250 mm internal diameter 1.7 mM CSH C18, analytical
column (Waters, UK). A 1 ml aliquot of the sample was transferred
to a 5 ml loop and loaded on to the column at a flow of 300 nl/min
for 5 minutes at 5% B. The loop was then taken out of line and the
flow was reduced from 300 nl/min to 200nl/min in 0.5 minute.
Peptides were separated using a gradient that went from 5% to 18%
B in 34.5 minutes, then from 18% to 27% B in 8 minutes and finally
from 27% B to 60% B in 1 minute. The column is washed at 60% B
for 3 minutes before re-equilibration to 5% B in 1 minute. At 55
minutes the flow is increased to 300 nl/min until the end of the run
at 60 minutes. Mass spectrometry data were acquired in a data-
directed manner for 60 minutes in positive mode. Peptides were
selected for fragmentation automaticallybydata-dependent
analysis on a basis of the top 12 peptides with m/z between 300
to 1750 Th and a charge state of 2, 3 or 4 with a dynamic exclusion
set at 15 seconds. The MS Resolution was set at 120,000 with an
AGC target of 3 x10
6
and a maximum fill time set at 20 ms. The
MS2 Resolution was set to 30,000, with an AGC target of 2 ×10
5
,a
maximum fill time of 45 ms, isolation window of 1.3 Th and a
collision energy of 28. All data were collected in centroid mode. Raw
files were then converted to mascot generic format (.MGF) files,
which were searched against a locally curated database
(Supplementary Table S4) of elasmobranch collagen type 1
(COL1A1 and COL1A2) and type 2 (COL2A1) sequences
obtained from the protein BLAST search of the elephant shark
(Callorhinchus milii). To assist evaluation of the manually selected
peptide biomarkers, the resultant.MGF files for each Error Tolerant
search were combined, filtered by removing peptides of <10 ion
score, and sorted via m/z value.
Results
Peptide composition of the mass
spectrometric fingerprints
The results indicate that, despite additional complexity of the
collagen peptide mass fingerprint obtained due to the presence of
the cartilaginous type II collagen, ZooMS provides exceptionally
high taxonomic resolution in this group, yielding species-level
identifications. Although only three of the species included in this
study had known collagen type I (COL1A1 and COL1A2) and type
II (COL2A1) sequences, Hypanus sabinus, Pristis pectinata and
Carcharodon carcharias (see Supplementary Tables S5–S7
respectively for LC-MS/MS sequencing results), it was clear that a
large proportion of peptides in the fingerprints derived from
COL2A1, both in the archaeological (Figure 2) and the modern
samples (Supplementary Figures S1,S2). Although differences in
relative abundance of particular peaks could be observed in some of
our peptide mass fingerprint comparisons between each sampled
modern tissue type (e.g., tooth vs. vertebrae; Supplementary Figures
S3–S5), they were not unexpected, reflecting the difference in
dominant protein, whether COL1 or COL2.
Taxonomic resolution: modern
comparative baseline
Species-level differences between the collagen peptide mass
fingerprints for modern comparative specimens could readily be
observed (Supplementary Figures S6–S16), even for each one of the
12 species of Carcharhinus and the four species of Sphyrna analyzed
here (Table 3). Sampling of the two Pristis species of relevance to
this study showed that the initially supplied reference material from
P. pristis was incorrect, deriving from P. pectinata, more in keeping
with geographical origins of the reference material. These
differences, proposed as species biomarkers (Supplementary Table
S8;Supplementary Figures S17–S51), were clearly visible in the
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peptide mass fingerprints of the archaeological specimens
also (Figure 3).
Taxonomic resolution:
zooarchaeological baseline
Although just over 30% (n=41) of the 137 analyzed
archaeological samples did not yield suitable quality collagen
fingerprints, the 98 specimens that did covered a greater than
expected range of taxonomic diversity based on morphological
identification, with 12 distinct groups identified (Figure 4). While
the majority of these results derived from specimens that could
not be morphologically identified below the family level
(i.e., Carcharhinidae) and often order level, 48 of our samples did
derive from a genus-level or lower suggested identification, though
mostly (n=33) from the hammerheads Sphyrna spp.
(Supplementary Table S3); of the 40 of these that were successful
(7 of the 8 poor collagen samples were of Sphyrna, with one from
Galeocerdo), 10 were initially incorrect on the original
morphological identification, and one of these (#78) cautioned/
corrected prior to ZooMS analysis (initially considered
Rhizoprionodon terraenovae and later suggested on morphological
grounds as potentially deriving from hammerhead [Sphryna]), an
identification confirmed by ZooMS and improved to the level of the
bonnethead (S. tiburo). All other misidentifications were from
specimens morphologically identified as hammerhead, corrected
to Atlantic sharpnose shark (R. terraenovae) in six instances (#70,
87, 99, 101, 103, 110), the lemon shark (Negaprion brevirostris)in
two instances (#66, 95), and the blacktip shark (C. limbatus) in one
instance (#61). These results are suggestive of misleading
morphological criteria used between the two taxonomic groups
during initial identification. However, we also note the value in
morphological identification, which remains important given the
substantial number of specimens that do not yield collagen/
biomolecular information, which, in this case comprised as much
as approximately one third of the samples analyzed in this study,
and is likely to have taphonomic biases that relate to species
and element.
Consistent with the morphological identifications, the most
abundant shark taxon observed via ZooMS among the Clupper
specimens was Carcharhinidae (i.e., migratory live-bearing sharks
of warm seas). Within this family, the lemon shark was the most
frequently identified among the sampled specimens. There are only
two species of lemon shark within the genus Negaprion. The lemon
shark (N. brevirostris) inhabits oceanic waters of the Americas, and
the sicklefin lemon shark (N. acutidens) inhabits the Indo-Pacific.
Lemon sharks prefer shallow coastal waters, often feeding at night
(Wetherbee et al., 1990) and growing to >3 m in length (Dibattista
et al., 2007). Lemon sharks are abundant in the Keys and are known
to inhabit inshore marine habitats, such as coral reefs or flats, as well
as estuaries (Compagno, 1984;Jennings et al., 2008;Tinari and
Hammerschlag, 2021;Wiley and Simpfendorfer, 2007).
Carcharhinus is the largest genus of the Carcharhinidae family,
containing at least 35 different species. Of those identified at Clupper,
the finetooth shark (C. isodon) is commonly found in the western
Atlantic from North Carolina to Brazil. Relatively small (1.6-1.7 m)
and fast swimming, it forms large schools in shallow inshore coastal
FIGURE 2
Sequence-annotated MALDI peptide mass fingerprint (top spectrum) of dominant proteins extracted from a confidently identified archaeological
Hypanus sabinus vertebra (black text = COL1A1, red text = COL1A2, blue text = COL2A1; matched to peptides listed in Supplementary Table S5) with
zoomed in sections (A–D) in order of increasing m/z.
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waters and habitats (Compagno, 1984). It is known to migrate
seasonally to follow warm waters (Castro, 1993), including those
surrounding Florida during the winter. Also identified within the
Clupper assemblage, the blacktip shark (C. limbatus) has a worldwide
distribution in tropical waters and is usually found in groups of
varying size in waters less than 30 m deep, including mangrove and
estuary habitats, as well as favoring island lagoons and drop-offs near
coral reefs (Castro, 1996). The blacktip is described as a stocky
species, typically reaching between 1.2 to 1.9 m, and only rarely
reaching 2 m in length (Castro, 1996;Compagno, 1984). While this
species is present in waters of the Gulf of Mexico, southern Florida
and the Keys year-round (Tinari and Hammerschlag, 2021;Wiley
and Simpfendorfer, 2007), it tends to be most abundant during winter
months after migrating southward from nursery grounds along the
northern Gulf coast, Carolinas, and Georgia (Castro, 1996;Heithaus
et al., 2007). It occurs in inshore habitats with shallower waters. The
dusky shark (C. obscurus) is one of the largest members of this genus,
at 3-4 m in adult length; it spends most of its time at 10-80 m depths
and within the western Atlantic migrates south during the winter
(Natanson and Kohler, 1996).
The Atlantic sharpnose shark (R. terraenovae), the third most
identified shark in this study, is also a relatively small shark (~1 m
adult length) (Compagno, 1984). It is one of the most frequently
encountered sharks in subtropical waters off the north-western
Atlantic Ocean today, including Florida, and the Gulf of Mexico
(Branstetter, 1990;Marquez-Farias and Castillo-Geniz, 1998;
Parsons and Hoffmayer, 2005). It is considered a coastal species
that often engages in regular inshore and offshore migration to
depths of up to 280 m (Compagno, 1984). It is found in a variety of
habitats such as shallow seagrass beds to deep non-vegetated sand
or mud and utilizes a series of coastal bays and estuaries as nurseries
for juvenile development (Carlson et al., 2008). Atlantic sharpnose
sharks are year-round residents of the Gulf of Mexico, Florida and
the Keys, but are most common during the winter (Tinari and
Hammerschlag, 2021); they are similar in length to bonnetheads,
with the morphology of the vertebrae being especially difficult to
distinguish between the two species.
The tiger shark (Galeocerdo cuvier) is the only extant species in
the family Galeocerdonidae. It is a large (>5 m), solitary, mostly
nocturnal hunter, moving closer to shore to feed (Compagno,
1984). It inhabits coastal and open ocean environments, and is
highly mobile, engaging in ontogenetic and seasonal migrations
(Ajemian et al., 2020;Lea et al., 2015). It prefers tropical waters
during the winter (e.g., Caribbean and Florida) and moves to high-
FIGURE 3
Example peptide mass fingerprints showing taxonomic resolution amongst archaeological samples of carcharhinids; central portion shows the most
diagnostic regions (A–D) for species determination of the mass spectra for each of the four elasmobranches Carcharinus isodon (orange; top), C.
limbatus (blue; second to top), Negaprion brevirostris (red; second to bottom) and C. obscurus (green; bottom) surrounded by zoomed in spectra
(A–D) of increasing m/z.
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latitude oceanic areas in the summer (Lea et al., 2015). Although it is
known to inhabit a variety of inshore habitats, such as estuaries,
reefs, and lagoons (Compagno, 1984), there is also an increased use
of continental-slope and deep-water habitats with increasing size
(Ajemian et al., 2020). Off the coast of Florida, it is most often found
in deeper oceanic waters (Tinari and Hammerschlag, 2021). The
nurse shark (Ginglymostoma cirratum) is the only species of shark
from a different order than ground sharks, the Orectolobiformes. It
is a very common inshore, bottom-dwelling shark, often inhabiting
waters of one meter or less deep and up to 12 m. It is typically ~3 m
in size and is found on rocky reefs, channels between mangrove
keys, and sand flats (Compagno, 1984). It is the most common
shark found year-round in the shallow waters of the Gulf of Mexico,
Florida, and the Keys (Heithaus et al., 2007;Tinari and
Hammerschlag, 2021;Wiley and Simpfendorfer, 2007). Nurse
sharks are largely nocturnal, often described as sluggish during
the daytime, and frequently found aggregating in large sedentary
resting groups (Compagno, 1984).
After the requiem sharks mentioned above, the second most
frequently identified shark taxonomic group was the hammerheads,
of the genus Sphyrna. Hammerheads are common, coastal inshore
sharks among the Keys, and like most sharks, they are solitary
hunters in the evening. The hammerhead species identified from
Clupper were the bonnethead (S. tiburo)andscalloped
hammerhead (S. lewini). The bonnethead is the smallest of
hammerhead species, typically <1 m in length, but can reach up
to 1.3 m in size (Carlson and Parsons, 1997;Compagno, 1984). This
species prefers inshore, coastal habitats such as seagrass beds,
FIGURE 4
Pie charts showing (A) species composition of archaeological remains analyzed by ZooMS, (B) the ZooMS results of species identified
morphologically as Carchariniformes, and (C) the ZooMS results of species identified morphologically as Sphyrna (bottom right).
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shallow bays, and estuaries primarily ranging from 10-25 m in
depth (Compagno, 1984;Heupel et al., 2006). In fact, this species
tends to be a long-term resident of these environments (e.g., Pine
Island Sound, Florida Bay, and Lower Florida Keys) with some
residents not undertaking long coastal migrations (Heithaus et al.,
2007;Heupel et al., 2006;Wiley and Simpfendorfer, 2007).
Although bonnetheads do not engage in true schooling, they are
known to aggregate together frequently (Compagno, 1984). The
scalloped hammerhead is a large hammerhead species, reaching a
maximum size of about 3.7 to 4 m in length (Compagno, 1984). It
frequents coastal warm temperate and tropical seas, alternating
between coastal and pelagic phases. It can be found over continental
and insular shelves (<200 m depth) and in adjacent deep waters
(Gallagher et al., 2014;Wells et al., 2018). The scalloped
hammerhead also frequents shallow bays and estuaries, with
nurseries typically located inshore (Corgos & Rosende-Pereiro,
2022). It is considered partly migratory and highly mobile with
natural reefs and hard-bottom outcroppings being important
foraging areas (Compagno, 1984;Wells et al., 2018). Scalloped
hammerheads are the only species of large-bodied shark that
engages in highly organized complex social schooling behavior
(Gallagher et al., 2014;Klimley, 1985;Klimley and Nelson, 1981).
With regards to the batoids, three species were identified in this
study, with a possible unconfirmed fourth (#18). There were at least
two rays of the order Myliobatiformes, including the Atlantic
stingray (Hypanus sabinus) and the spotted eagle ray (Aetobatus
narinari), which is the only species of its genus found in the Atlantic
(Richards et al., 2009). They can both be found regularly in the
western Atlantic Ocean, including the Keys, but with different
habitat preferences. The Atlantic stingray can reside in low
salinity and has a high tolerance for shallow estuarine and
freshwater habitats; spotted eagle rays are known to occur in
inshore marine waters, including reef and mangrove habitats, but
the species largely prefers open waters (DeGroot et al., 2021).
The smalltooth sawfish (Pristis pectinata) was the third identified
batoid of the order Rhinopristiformes from among the analyzed
specimens. While once found on both sides of the Atlantic Ocean
(e.g., Bigelow and Schroeder, 1953), this species is now restricted to
waters surrounding Florida, particularly within the Everglades
National Park, and the Bahamas (Carlson et al., 2013;
Simpfendorfer and Wiley, 2005;Wiley and Simpfendorfer, 2007).
The species is present all year in south Florida coastal waters, and
individual animal size tendsto correlate with habitat preferences; with
small individuals or juveniles observed in shallow coastal habitats,
such as mangroves and estuaries, while larger juveniles or adults are
observed among deeper or open waters (Waters et al., 2014).
Discussion
Mitigating limitations in identification
Given the challenges of elasmobranch identification in
zooarchaeology (e.g., Gilson and Lessa, 2021;Kozuch and
Fitzgerald, 1989;Prieto, 2023), and especially within tropical or
subtropical environments with high biodiversity such as the Keys,
the results of our analysis with the Clupper specimens are an
exemplar of the methodological promise that ZooMS holds for
improving elasmobranch species identifications across several
world regions and taxa. From the perspective of taxonomic
diversity and overcoming limitations in zooarchaeological
identification via ZooMS, there are now at least nine different
confirmed (including corrected identifications) shark species, two
stingray species, and one sawfish species documented for the site.
The high species-level taxonomic resolution achieved, for the first
time, has implications for advancing archaeological and historical
ecology research in the Keys and elsewhere.
Implications for Ancestral Indigenous
harvest and elasmobranch historical
ecology in the Keys
When considered within the context of contemporary
elasmobranch diversity and distribution, the results provide a new
opportunity to think beyond generalities gleaned from order- or
family-level identifications and information (e.g., LeFebvre et al.,
2022;Oliveira, 2024). Here we focus on contemporary species
occurrence and co-occurrence observations from the Keys as a
foundation for deeper interrogation of elasmobranch harvest and
historical ecology represented by the Clupper data.
As part of a recent study investigating shark assemblages
spanning state and federal management areas from Miami into
the middle Keys (including Upper Matecumbe Key), Tinari and
Hammerschlag (2021) (see also Table 1) assessed relationships
between species occurrences and abundances by habitat type (i.e.,
Bay or Ocean), depth, and season (i.e., wet season from May-
October and dry season from November-April). They also
considered correlations between species size and occurrence. The
authors found that while shark assemblages within the study region
are characterized by year-round occurrences for the majority of
observed species [see Table 1 in Tinari and Hammerschlag (2021)],
there are also species-level nuances to occurrences. For example,
nurse, tiger, and Atlantic sharpnose sharks have higher probabilities
of occurrence in offshore habitats (i.e., ocean), compared to blacktip
and lemon sharks found in inshore habitats (i.e., bay). Also, while
temperature is not a significant driver of shark occurrence, salinity
is for two of the most common species in the Keys, i.e., nurse sharks
(i.e., decrease in occurrences with increases in salinity) and lemon
sharks (i.e., increase in occurrences with increases in salinity).
Depth is also a factor; tiger sharks occur in deeper waters, and
nurse, blacktip, and lemon are found in shallower waters. Finally,
there are variable correlations between some species sizes (i.e., total
length) and season and habitat; whereby, occurrences of larger
nurse and bull sharks are higher in the Keys, usually in offshore
waters, during the dry season compared to Miami, larger lemon
sharks have relatively higher occurrence in offshore habitats overall,
and larger Atlantic sharpnose sharks occur more frequently in the
Keys compared to waters near Miami.
Like species occurrences, fishery species co-occurrences and
community composition are usually related to overlapping habitat
preferences and dietary habits, both of which may vary throughout
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a day (e.g., nocturnal versus diurnal feeding) and/or season, as well
as across sex, age, and reproductive cycles of different species
(Heithaus et al., 2007). For the Keys and most germane to this
study, nurse and bonnethead sharks are among the more frequently
observed sharks in the Keys today and they have high rates of co-
occurrence with several other elasmobranch species, including an
inshore habitat overlap with sharpnose and lemon sharks, as well as
with the southern and white-spotted eagle rays (Ramey, 2021).
Assuming contemporary patterns in elasmobranch species
occurrence and co-occurrence, as well as the current
environmental conditions of the Keys and surrounding waters,
extend into the deep-past, our species-level results from the
Clupper site suggest that several of the sharks and rays
represented would have been opportunistically, and/or
predictably, available for harvest from the same habitats as part of
a generalist, primarily inshore, fishery at Clupper (e.g., LeFebvre
et al., 2022;Oliveira, 2024). Common among inshore, shallow water
(e.g., Bay) habitats, we can hypothesize that species, such as lemon,
nurse, blacktip, and bonnethead sharks, were the most commonly
harvested species based on their likely proximity to the shorelines of
Upper Matecumbe Key and that their smaller overall body size was
amenable to net and trap capture. We can also hypothesize that co-
occurrences between elasmobranch and bony fishes also likely
shaped the fishery given that the most frequently identified bony
fish taxa from Clupper include catfishes (Ariopsis felis and Bagre
marinus), grunts (Haemulon spp.), jacks (Caranx spp.), snappers
(Lutjanus spp.), and groupers (Epinephelus spp. and Mycteroperca
spp.) - all of which are commonly encountered among the same
inshore habitats as the identified elasmobranch species. Species
dietary patterns also have implications for hypotheses regarding
elasmobranch harvest. For example, scalloped hammerhead, tiger,
and lemon sharks are known to feed on bony fishes (e.g., jacks,
catfishes), sea turtles, rays, and/or relatively smaller sharks (e.g.,
blacktip shark, nurse shark) within inshore habitats (e.g.,
Compagno, 1984), all of which are represented within the
Clupper zooarchaeological record.
However, these hypotheses, largely based on co-occurrence
inferences, do not necessarily preclude possible offshore or
deeper-water harvest practices at Clupper that aimed to target
more pelagic species. Three of the largest-growing elasmobranch
species identified among the study specimens, the dusky shark,
scalloped hammerhead, and white-spotted eagle ray, are also known
to inhabit deeper, offshore waters as adults, depending on the time
of day, seasonal migrations, and reproduction habits. This is similar
to the largest-growing bony fish identified from Clupper, the sailfish
(Istiophorus platypterus)(Oliveira, 2024). These taxa may have been
harvested while crossing between offshore (i.e., ocean) and inshore
(i.e., bay) habitats of the Keys. The Clupper site is located adjacent
to a natural deep-water pass that would have made such
crossings possible.
We can also begin to think more critically about technological
approaches to elasmobranch harvest at Clupper, all of which would
have been informed by traditional ecological knowledge developed
across generations of cultural practices at the village and more
broadly across the Keys (Oliveira, 2024). Approaches likely
included several types of gear, including multi-species harvest
techniques, such as nets and/or traps intended to capture a
diversity of species at once (e.g., elasmobranchs and bony fishes),
or those intended for one individual at a time, such as single hook
and line and/or spear (Walker 2000). For example, contemporary
commercial and recreational fisheries data show that finetooth
sharks may be efficiently harvested using gillnets (Portnoy et al.,
2016), while blacktip, dusky, and bonnethead sharks are captured
using baited hook and line (e.g., individual angling or longline) (e.g.,
Ulrich et al., 2007). Species such as nurse sharks are also known to
take bait intended for other fishes and raid fish nets or traps (Castro,
2000). Testing these ideas and hypotheses will require larger sample
sizes identified to species for the calculation of relative abundances,
as well as measurements of individual sizes and approximate ages
represented across taxa.
From a geographic perspective of contemporary species
occurrences, all the elasmobranch species identified from
Clupper, except for finetooth shark, are known to inhabit the
Keys today. The finetooth shark is not regarded as common in
the Keys based on contemporary survey data of species distribution.
While the finetooth shark has long been known to inhabit the
waters off the peninsular coast of Florida during the fall and into
winter (Castro, 1993), there was in 2007 the report of a new
southern extent of the species occurrence to include Florida Bay
waters along the southern terminus of peninsular Florida (Wiley
and Simpfendorfer, 2007), well beyond the previously documented
southern extent of the species around Lemon Bay on the southwest
coast and Port Salerno on southeast coast of the peninsula; the
authors reported that the presence of finetooth sharks in Florida Bay
was likely rare overall, but significant from the perspective of
possible exchange between Gulf and Atlantic stocks and learning
more about the species’seasonal migration movements and
relationship to water temperature. The species identification of
finetooth shark via ZooMS at Clupper may have several
implications from historical ecological and archaeological
perspectives, including: 1) finetooth sharks were more common in
the island region and/or greater Florida Bay in the past; 2) there is a
potentially deeper history for stock exchanges through time; and 3)
afinetooth shark, or portion of an individual, was transported to the
Clupper site post mortem from a more northern location along the
Florida peninsula. It could also indicate sea temperatures in the past
implying environmental/climatic change. To test these possibilities,
it will be imperative to identify more finetooth shark specimens
within the Clupper zooarchaeological assemblage, from additional
zooarchaeological assemblages from the greater Keys region, and
ideally from across southern portions of peninsular Florida that are
dated to the Ancestral Period.
In summary, we now have a more taxonomically refined and
accurate understanding of the elasmobranch diversity represented
at Clupper than we did prior to the ZooMS analysis, presenting an
opportunity to think beyond generalities gleaned from primarily
order- or family-level identifications and information. The data
indicate that inshore, and likely to a lesser extent offshore, habitats
local to or near the site supported a taxonomically diverse
elasmobranch fishery ca. AD 1,000-1,300. It is reasonable to
assert that elasmobranch harvest may have been linked to the
harvest of other taxa (e.g., bony fishes and sea turtles) and
Buckley et al. 10.3389/fmars.2024.1500595
Frontiers in Marine Science frontiersin.org13

facilitated using multi-species capture nets and/or traps. This may
have been most prevalent among smaller species, such as finetooth
sharks, blacktip sharks, and bonnetheads in shallower-water
habitats. Multi-species capture methods may have also provided
opportunities for opportunistic spear fishing of elasmobranchs
attracted to fishes caught in nets or traps, such as nurse or bull
sharks. Concomitant with inshore harvest and variable uses of
fishery technology, some of the species represented may have also
been pursued through more targeted approaches, such as hook and
line, following seasonal availability depending on aggregation (e.g.,
S. lewini and S. tiburo) and/or migration habits across both inshore
and offshore waters, including larger, migrating species more
abundant within the Keys during the winter months (e.g.,
hammerhead and tiger sharks).
Implications for future zooarchaeological
research and supporting conservation
The application of ZooMS to the study of elasmobranchs has
potential for improved historical ecological baselines derived from
zooarchaeological assemblages, and particularly in support of
conservation. Because marine resource managers, conservation
practitioners, and fisheries scientists often work with species-level
data, the results offer an opportunity to consider how to better leverage
zooarchaeological analyses and data to support species-specificaswell
as community-level historical baselines of elasmobranch diversity and
distribution in the Keys (e.g., Tinari and Hammerschlag, 2021), albeit
through a cultural lens of human selection and post-depositional
taphonomic processes (e.g., preservation). Nonetheless, the species
identified among the Clupper specimens are now known to have a
deep-time history of human engagement and harvest, pre-dating 20
th
and 21
st
centuries scales of impact. Approaching the Clupper
zooarchaeological species list as a historical survey, similar to more
recent survey lists (e.g., Ramey, 2021;Tinari and Hammerschlag, 2021;
see also Table 2), but providing a baseline of species occurrences well
before the precipitous losses of recenthistory, we suggest three lines of
possible future research, aimed at elucidating trends (e.g., stasis or
change) in species diversity through time, across space, and among
groups of people (i.e., cultural practices).
1. Given that almost all identified species from Clupper are
extant, we can target specific species for genetic analyses
leveraging aDNA (e.g., Shepherd and Campbell, 2021), and
ultimately having the potential to contribute to critically
needed long-term phylogenetic perspectives of
elasmobranch species diversity and loss through time.
The one as yet unidentified ray leaves open the potential
for new species discovery, at least likely locally extinct.
2. With the use of high-throughput digital scanning techniques
linked with ZooMS (e.g., Buckley et al., 2021), we can
compare fragmented, species-identified elasmobranch
zooarchaeological specimens with modern comparative
specimens for the possible identification of species-specific
morphological hallmarks not previously recognized.
3. Because elasmobranch species occurrences can be highly
variable in terms of individual age and size within a given
habitat, depending on many factors (e.g., nursery locations,
water depth, salinity, adult migration patterns), and
particularly within habitat-diverse tropical locations such
as the Keys, we can begin to leverage ZooMS-identified
specimens, including bulk-assemblage approaches (e.g.,
Buckley et al., 2016;Oldfield et al., 2024), to link specimen
identification with allometric size data across species (e.g.,
species length and age correlations). These data would allow
us to measure species sizes (and approximate ages),
represented within the zooarchaeological record, and
quantify relative species abundances accordingly (e.g.,
MNI, NISP), ultimately contributing to deep-time
perspectives of species sizes beyond more recent
time scales (e.g., decadal) as well as more specific
interpretations of elasmobranch harvest habitats and
practices in the past.
Not surprisingly, difficulties surrounding species-level
elasmobranch identification of zooarchaeological specimens can
significantly limit the information that can be garnered from
preserved bone, teeth, and denticles, including insights into
possible habitats of harvest and capture methods used in the past.
Furthermore, identifications only to order or family level seriously
diminish the historical ecological implications that can be drawn,
which often necessitate species-level resolution and inference to
support conservation and understanding the impacts of human
predation through time (e.g., harvest for subsistence, shark fin
markets). Archaeological elasmobranch assemblages, such as
Clupper, are composed of specimens that hold critical potential to
constructing as taxonomically, geographically, and temporally as
possible baselines of diversity through time, and particularly within
the context of long-term human engagement. The species identified
from Clupper include several taxa considered to be vulnerable,
endangered, or critically endangered (Table 1), particularly those
characterized by K-selected traits, making them incredibly
vulnerable to loss as noted earlier. While the use of ZooMS in
establishing robust species-level identifications of elasmobranchs
has been successful, its future use will allow us to answer many more
questions and develop methodological advances that are not
possible based on morphological identifications alone, perhaps
most suitably to applications involving wildlife forensics,
including the population-decimating shark fin trade (Cardeñosa
et al., 2022). However, we assert that both ZooMS and morphology
are integral to realizing the full potential of zooarchaeological data
to contribute to conservation baselines and deep-time historical
perspectives in the Keys and beyond.
Data availability statement
The original contributions presented in the study are available
on FigShare at https://figshare.com/articles/dataset/Clupper_
elasmobranch_species_identification_using_ZooMS/27087190?
Buckley et al. 10.3389/fmars.2024.1500595
Frontiers in Marine Science frontiersin.org14

file=49358374 and presented as additional figures in the
Supplementary Material, further inquiries can be directed to the
corresponding author/s.
Ethics statement
Ethical approval was not required for the study involving animals
in accordance with the local legislation and institutional requirements
because deceased animal tissues from museum collections were used.
Author contributions
MB: Conceptualization, Data curation, Formal analysis,
Funding acquisition, Investigation, Methodology, Project
administration, Resources, Supervision, Validation, Visualization,
Writing –original draft, Writing –review & editing. E-MO: Data
curation, Formal analysis, Investigation, Methodology, Writing –
review & editing. CO: Data curation, Formal analysis, Methodology,
Validation, Visualization, Writing –original draft. CB: Data
curation, Formal analysis, Visualization, Writing –original draft.
AK: Resources, Writing –original draft. NF: Resources, Writing –
review & editing. TA: Resources, Writing –review & editing. VT:
Writing –review & editing. SF: Writing –review & editing. ML:
Resources, Writing –original draft, Writing –review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. Funding for
excavation was provided by the University of Miami, University of
Oregon, and University of Georgia. Zooarchaeological analysis was
funded by the Florida Museum of Natural History John S. and
James L. Knight Professorship in Archaeology; the PhD studentship
for Oldfield was funded for by the BBSRC. The BBSRC funded the
PhD studentship for Ellie-May Oldfield, grant number BB/
T008725/1.
Acknowledgments
From the Florida Museum of Natural History, we thank Kitty
Emery for her support in the use of the Environmental Archaeology
Program modern comparative skeletal collection, Rob Robins and
Gavin Naylor for their guidance on taxonomy, and Coleman Sheehy
for leading compliance with CITES regulations for international
studies. We also thank Laura Kozuch and Simon-Pierre Gilson for
the invitation to share some of this work at the 2024 Society for
American Archaeology Annual Meeting. The Clupper
archaeological research was conducted under permit 1415.015 to
Thompson from the Division of Historical Resources, Florida
Department of State (2014) permit 11051415 to Thompson from
the Florida Park Service, Florida Department of Environmental
Protection (2015). The authors thank avocational archaeologist Jim
Clupper, after whom the site is named, for invaluable assistance
with this research.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fmars.2024.1500595/
full#supplementary-material
SUPPLEMENTARY FIGURE 1
Sequence-anno tated MALDI peptide mass fingerprint (top) of dominant
proteins extracted from modern Hypanus sabinus tissue (black text =
COL1A1, red text = COL1A2, blue text = COL2A1; matched to peptides
listed in Supplementary Table S5) with zoomed in sections (A-D) in order of
increasing m/z.
SUPPLEMENTARY FIGURE 2
Sequence-anno tated MALDI peptide mass fingerprint (top) of dominant
proteins extracted from modern Carcharodon carcharias tissue (black text
= COL1A1, red text = COL1A2, blue text = COL2A1; matched to peptides listed
in Supplementary Table S5) with zoomed in sections (A-D) in order of
increasing m/z.
SUPPLEMENTARY FIGURE 3
MALDI spectra showing peptide mass fingerprints (top) of different modern
tissues (vertebral process cartilage and tooth) from modern Carcharhinus
limbatus, with zoomed in sections (A-C) in order of increasing m/z.
SUPPLEMENTARY FIGURE 4
MALDI spectra showing peptide mass fingerprints (center) of different
modern tissues (cranial cartilage and vertebra) from Ginglymostoma
cirratum, with zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 5
MALDI spectra showing peptide mass fingerprints (centre) of different
modern tissues (jaw, tooth and ‘cranial cartilage’) from Galeocerdo cuvier,
with zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 6
MALDI spectra showing peptide mass fingerprints (top) of modern
Carcharhinus isodo n, Carcharhinus limbatus, Carcharh inus signatus and
Carcharhinus leucas, with zoomed in sections (A-D) in order of increasing m/z.
Buckley et al. 10.3389/fmars.2024.1500595
Frontiers in Marine Science frontiersin.org15

SUPPLEMENTARY FIGURE 7
MALDI spectra showing peptide mass fingerprints (top) of modern Carcharhinus
galapagensis, Carcharhinus brevipinna, Carcharhinus obscurus and
Carcharhinus altimus,withzoomedinsections(A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 8
MALDI spectra showing peptide mass fingerprints (top) of modern
Carcharhinus perezi, Carcharhinus plumbeus, and Carcharhinus acronotus,
with zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 9
MALDI spectra showing peptide mass fingerprints (top) of modern Sphryna
lewini, Sphryna mokarran, Sphryna tiburo and Sphryna zygaena, with zoomed
in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 10
MALDI spectra showing peptide mass fingerprints (centre) of modern
Galeorhinus galeus, Carcharias taurus, Pristis pristis and Pristis pectinata,
with zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 11
MALDI spectra showing peptide mass fingerprints (top) of modern
Rhizoprionodon terraenovae, Mustelus canis, Alopias vulpinus and Isurus
oxyrinchus, with zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 12
MALDI spectra showing peptide mass fingerprints (centre) of Hexanchus
griseus,Negaprion brevirostris,Hemipristis elongata and Prionace glauca,
with zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 13
MALDI spectra showing peptide mass fingerprints (top) of modern
Bathytoshia centroura, Raja eglanteria and Aetobatus narinari, with zoomed
in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 14
MALDI spectra showing peptide mass fingerprints (top) of modern Rhinoptera
bonasus,Hypanus say, Urobatis jamaicensis and Hypanus americanus, with
zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 15
MALDI spectra showing peptide mass fingerprints (top) of remaining
archaeological vertebra examples; Aetobatus narin ari,Hypanus sabinus,
Pristis pristis and one from a confidently identified Hypanus sabinus barb,
with zoomed in sections (A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURE 16
MALDI spectra showing peptide mass fingerprints (top) of remaining
archaeological examples 2 –Galeocerdo cuvier, Ginglymostoma. cirratum,
Rhizoprionodon, Sphyrna tiburo and Sphyrna lewini, with zoomed in sections
(A-D) in order of increasing m/z.
SUPPLEMENTARY FIGURES 17–S51
LC-MS/MS spectra of peptides of potential interest as biomarkers.
SUPPLEMENTARY TABLE 1
Current zooarchaeological resul ts from the Clupper site midden across
vertebrate classes.
SUPPLEMENTARY TABLE 2
Modern reference specimens with details.
SUPPLEMENTARY TABLE 3
Archaeological specimens with details.
SUPPLEMENTARY TABLE 4
Local protein sequence database accession numbers.
SUPPLEMENTARY TABLE 5
COL1A1, COL1A2 and COL2A1 matches to Hypanus sabinus.
SUPPLEMENTARY TABLE 6
COL1A1, COL1A2 and COL2A1 matches to Carcharodon carcharias.
SUPPLEMENTARY TABLE 7
COL1A1, COL1A2 and COL2A1 matches to Pristis pectinata.
SUPPLEMENTARY TABLE 8
Selected peptide biomarkers.
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