The first amber caridean shrimp from Mexico reveals the ancient adaptation of the Palaemon to the mangrove estuary environment

The aquatic and semiaquatic invertebrates in fossiliferous amber have been reported, including taxa in a wide range of the subphylum Crustacea of Arthropoda. However, no caridean shrimp has been discovered so far in the world. The shrimp Palaemon aestuarius sp. nov. (Palaemonidae) preserved in amber from Chiapas, Mexico during Early Miocene (ca. 22.8 Ma) represents the first and the oldest amber caridean species. This finding suggests that the genus Palaemon has occupied Mexico at least since Early Miocene. In addition, the coexistence of the shrimp, a beetle larva, and a piece of residual leaf in the same amber supports the previous explanations for the Mexican amber depositional environment, in the tide-influenced mangrove estuary region.

a shrimp, a beetle larva and a piece of leaf, extends our understanding on the paleontological depositional environment. It is reconfirmed that the ancient environment of amber deposition in Mexico is a tidal affected mangrove estuary area.  (Figs 1a, b, 2 and S1). The materal deposited in the the Paleo-diary Museum of Natural History, Beijing, China.
Etymology: The specific name comes from the Latinization "estuary" where the shrimp inhabited. Remarks: P. aestuarius sp. nov. resembles Palaemon vesolensis Bravi, Coppa, Garassino & Patricelli, 1999, the difference between them in: P. aestuarius sp. nov. with nine teeth on the dorsal margin, carapace bearing prominent branchiostegal groove; but the P. vesolensis with seven dorsal teeth, carapace without traces of grooves 20 .
Description: Rostrum long, reaching the distal end of scaphocerite, slightly shorter than the carapace; basal crest absence, dorsal margin nearly straight, faintly convex in the middle; with nine teeth, including one tooth on carapace, large and sharp, tips forward; basal four teeth evenly distributed, the distance between them greater than the intervals of the other teeth.
Eyes well developed, cornea broader than stalk. Antennules triflagellate, scaphocerite slender, about five times as long as wide, outer margin nearly straight, basal area with one long narrow spiny projection, pointed forward. Carapace smooth, hepatic spine absent, without granular process; the branchiostegal groove extended longitudinally backward; branchiostegal spine sharp, situated on anterior margin of cephalon, pointed forward.
The apex of third maxillipede reaching the middle area of scaphocerite. First pereiopod slightly robust, carpus as long as merus, folding inward. The second pereiopod absent. Third pereiopod normal, ischium long, nearly length of propodus; merus longest, about three times as long as carpus; dactylus shortest, with merus ratio 1: 3.6. Forth pereiopod longer than the third pereiopod, ischium slightly longer than the length of propodus, about two times as long as carpus; dactylus shortest, merus longest, with ratio 1: 4.2. Fifth pereiopod longer than the forth pereiopod, ischium nearly equal length of propodus, about two times as long as carpus; dactylus shortest, merus longest, with ratio 1: 4.4.
Abdomen glabrous, with six segments, central uplift, pleurite of the second pleomere covering the pleurites of the first and third; ventral margin of the second pleurites with a central notch, first to third pleurites broadly rounded. Telson conical, 0.6 times as long as sixth abdominal segment; uropods long and narrow triangular, apices sharp, nearly three times the length of the telson.

Discussion
With the emergence of a variety of aquatic organisms, semiaquatic organisms and insects, the sedimentary paleoenvironment of Mexican amber is generally considered to be a coastal flood-plain suffering from tidal influence in mangrove estuary environment 16,18 . Affected by floods and tides, many ponds and small depressions formed in tidal flat and the surrounding land. Unique environmental conditions make Mexican amber possible to embed various inclusions, including aquatic and terrestrial organisms, particularly abundant crustaceans, relative to other famous amber localities such as Baltic, Dominica, and Myanmar etc. 18,19 . Although numerous aquatic and semi-aquatic groups of amber inclusions have been reported in Mexican amber, such as Copepoda, Ostracoda, Tanaidacea, Amphipoda, Isopoda and Brachyura, there are still more species groups to be discovered and studied 14 . The shrimp presented in this study is the first record of caridean species preserved in amber, adding an important dimension to the inclusion diversity of Mexican amber.
So far, the Palaemonoidea contains nine genera and 17 definite paleontological species, all of which are included in Palaemonidae, and preserved in rock sediments 12,21,22 . Four genera reported entirely in Cenozoic, Bechleja Houša, 1957, Propalaemon Woodward, 1903, Pseudocaridinella Martins-Neto & Mezzalira, 1991, Micropsalis Von Meyer, 1859, and we can rule this shrimp out of them by the following morphological features: with triflagellate antennule; having a rostrum with a single tip and lacking hepatic spine; bearing a serrate rostrum on dorsal margin; with a short abdomen and without significant chela on first pereiopod 21,23,24 . There are also four genera recorded in the Mesozoic completely. Alburnia Bravi & Garassino, 1998 possesses only one species found in Lower Cretaceous of Italy, which is significantly different from the P. aestuarius sp. nov. with ten teeth www.nature.com/scientificreports www.nature.com/scientificreports/ on dorsal rostrum, no traces of branchiostegal groove and branchiostegal spine 25 . Beurlenia Martins-Neto & Mezzalira, 1991 has one species preserved in Lower Cretaceous of Brazil, which is distinct from the P. aestuarius sp. nov. with antennal spine and 14 teeth on dorsal rostrum 26 . Schmelingia Schweigert, 2002 has a single species preserved in Upper Jurassic of Germany, which is different from the P. aestuarius sp. nov. with surface of shell with punctate lines and rostrum with one tooth at apex 27 . Yongjicaris Garassino, Yanbin, Schram & Taylor, 2002 has a single species preserved in Lower Cretaceous of China, which is different from the P. aestuarius sp. nov. with carapace without groove and spine, rostrum without teeth 28 . According to the diagnosis, the shrimp cannot belong to the above genera.
Although the juvenile shrimp in the amber is not well preserved and the most important second pereiopods are missing, we can still classify it into the genus Palaemon of Palaemoninae by the following characters: body slender, rostrum armed with non-movable teeth, without coronal projection in basal area, absence of hepatic spine, bearing distinct branchiostegal groove and branchiostegal spine 29 . According to the last two characters, we can separate it from the related genus Macrobrachium. In addition, compared to its sister lineage of Pontoniinae, which is strictly marine, with the individuals small and robust, and symbiotic with other marine organisms, Palaemoninae is distributed in a wide salt environment, mainly in shallow sea and freshwater, with individuals relatively thin and living freely. Therefore, we believe that the shrimp should belong to the Palaemon of Palaemoninae.
There are three definite fossil species belonging to the Palaemon, which are recorded in Europe from Cretaceous and Miocene 12,21,22 . In this study, we report the first Palaemon shrimp preserved in amber, which is the first trustworthy fossil Palaemon shrimp recorded in Mexico. Therefore, the origin of Palaemon can be traced back before the Early Cretaceous and had occupied Mexico at least since Early Miocene.
Seven species of Palaemon are known to exist in Mexico. We can distinguish the shrimp from other living species by the following characteristics: P. hobbsi with five to six teeth on dorsal margin and bearing distinct antennal spine 30 ; P. lindsayi with six or seven dorsal teeth, antennal spine sharp and antennal scale 3.5 times longer than wide 31 ; P. mexicanus with six to seven dorsal teeth, antennal spine sharp and distinct, scaphocerite about three times as long as wide, lateral margin slightly concave 32 ; P. mundusnovus with seven dorsal teeth 33 ; P. octaviae bearing ischium significantly shorter than the length of propodus in the last three pairs of pereiopods 34 ; P. paludosus with six to eight dorsal teeth 35 ; P. suttkusi with five to seven dorsal teeth, scaphocerite almost three times as long as wide, antennal spine strong 36 .
Efficient ability in osmotic pressure regulation enables Palaemon shrimps to adapt to the different saline-containing waters. The sedimentary environment of fossil species, recorded from Italy in Cretaceous and Miocene are coastal lagoons, and they share the same prominent feature of lacking branchiostegal groove and branchiostegal spine. This is consistent with the characteristics of the other families completely inhabiting the ocean in Palaemonoidea, including genera in Palaemoninae and all species in Pontoniinae. The shrimp in our study from mangrove estuary environment in Mexico during Early Miocene has branchiostegal groove and branchiostegal spine. Meanwhile, there are seven extant Palaemon species in Mexico, distributed in marine sandy mud flats, estuaries and freshwater, and all of them have branchiostegal groove and branchiostegal spine. In conclusion, branchiostegal groove and branchiostegal spine appeared gradually with the marine ancestor continuous invasion from the sea to freshwater, which is consistent with previous studies 37 . Furthermore, in the Early Miocene, there was already Palaemon shrimp living in freshwater or estuarine environment, which provided the possibility for further invasion to freshwater.
The most intriguing aspect about the amber is the inclusion of a shrimp together with a beetle larva, which may be a carabid, a staphylinid or a cucujid. The amber is thus so unusual that we try to reconstruct its formation process. During the rainy season in the southeastern Mexico, the weather was hot and humid. P. aestuarius sp. nov. lived in mangrove estuary environment, like many other crustaceans found in Mexican amber 16 , tides carrying it with a juvenile coastal beetle rushed to the tidal flat and farther land with the water level rising dramatically. Eventually, they were washed to the edge of a pond surrounded by amber trees, and the shrimp's athletic ability decreased significantly due to the loss of the second pereiopods during the flushing of the water. More unfortunately, the resin flow was so large that they were wrapped together. Alternative explanation is that the beetle larva clinging to the bark of amber tree was wrapped in flowing resin, and the resin stream captured the shrimp brought by tidal forces at the same time. The resin block continued to grow and flow into the water. When the rainy season passed, the water level of the pond gradually declined and eventually dried up, and the resin was fully solidified and dried. In addition, it is worth noting that we cannot completely rule out the possibility that the shrimp had adapted to freshwater. If so, it is likely that in a flood event, P. aestuarius sp. nov. rushed out of the riverbed and fell into a pond. When the pond dried up, the shrimp died, and then the resin flow wrapped it and the beetle larvae. After tens of millions of years of geological action, we finally found them in a piece of amber. In conclusion, the coexistence of the shrimp, a beetle larva, and a piece of residual leaf supports the previous explanations for the Mexican amber depositional environment, a coastal flood-plain affected by tides, rivers and streams, forming a mixed biome of brackishwater, freshwater and terrestrial arthropods 16,38 . Meanwhile, the discovery of P. aestuarius sp. nov. provides insight into early shrimp evolution and distribution, and sheds light on rich biodiversity in Mexican amber during the Early Miocene.