New Fossil Scorpion from the Chiapas Amber Lagerstätte

A new species of scorpion is described based on a rare entire adult male preserved in a cloudy amber from Miocene rocks in the Chiapas Highlands, south of Mexico. The amber-bearing beds in Chiapas constitute a Conservation Lagerstätte with outstanding organic preservation inside plant resin. The new species is diagnosed as having putative characters that largely correspond with the genus Tityus Koch, 1836 (Scorpiones, Buthidae). Accordingly, it is now referred to as Tityus apozonalli sp. nov. Its previously unclear phylogenetic relationship among fossil taxa of the family Buthidae from both Dominican and Mexican amber is also examined herein. Preliminarily results indicate a basal condition of T. apozonalli regarding to Tityus geratus Santiago-Blay and Poinar, 1988, Tityus (Brazilotityus) hartkorni Lourenço, 2009, and Tityus azari Lourenço, 2013 from Dominican amber, as was Tityus (Brazilotityus) knodeli Lourenço, 2014 from Mexican amber. Its close relationships with extant Neotropic Tityus-like subclades such as ‘Tityus clathratus’ and the subgenus Tityus (Archaeotityus) are also discussed. This new taxon adds to the knowledge of New World scorpions from the Miocene that are rarely found trapped in amber.


Introduction
The fossil record of the order Scorpiones Koch, 1851 is currently comprised of 118 described species, including 21 species from amber deposits worldwide [1][2] [3]. A comprehensive list of fossil scorpions has been recently given elsewhere [2]. Fossil amber scorpions have been consistently found since the Early Cretaceous. The oldest amber scorpion is Archaeobuthus estephani Lourenço, 2001 (family Archaeobuthidae), from the Early Cretaceous amber of Lebanon [4]. Preliminarily, A. estephani was considered a member of the superfamily Buthoidea Koch, 1837 [4]; as was Palaeoburmesebuthus grimaldii Lourenço, 2002 (family Incertae sedis) from mid-plesiomorphic characters seen in Tityus scorpions [23]. It also shares some ecological traits with groups that inhabit Neotropical forests. The phylogenetic position of T. apozonalli among fossil members of the family Buthidae from the Dominican Republic and Mexican amber sites is preliminarily examined herein. As a complement, the fossil record of the family Buthidae since the Cenozoic and extinct close relatives in the Mesozoic is also briefly reviewed.

On the fossil occurrence
The fossil studied here was collected in a hand-made pit at the Guadalupe Victoria site near Simojovel, Chiapas, south of Mexico (Fig 1), which comprises two indigenous settlements: The Guadalupe Victoria I and II [24] [25]. The crude amber piece was found by a native local farmer, who informed Director B. Luna of the Museo del Ámbar in San Cristobal de Las Casas, Chiapas, of his discovery. According to geographical references provided by the collector, a provenance analysis has been carried out in amber with infrared and X-ray spectroscopies. This analysis has been complemented with field geology tracking the outcrop at the amber site commonly known as the Guadalupe Victoria site [24] [25]. The present amber section consists of organic-rich lignite embedded in non-fissile, coarse to fine grained sandstone with variable thickness. A ripple cross-lamination, interbedded friable shales, stratified bentonite-like clay, copious brown to red iron oxides, pyrite and other secondary clay minerals were also observed. The organic-rich lignite is the result of plant decay and soil lithogenesis. The amber section overlies a transitional- marine sequence with abundant shells, corals, calcareous debris, bioclastic limestones and consolidated sandstones. The geological setting of the Simojovel area displays rhythmic sequences that are the result of sea level fluctuations. This indicates land changes from transitional to terrestrial environments and associated climate fluctuations that span from the Oligocene-Miocene boundaries to mid-Pliocene [26]. Consequently, the lithology and sedimentary record of the amber section is consistent with a terrestrial-fluvial environment near a coastal plain [27]. Palynology in the amber strata also suggests an ancient terrestrial-mangrove environment [28].
The available published paleontological and biostratigraphic data in the Simojovel area supports that amber-bearing beds are part of the Mazantic shale and Balumtum sandstone strata with a relative age estimated in the Early to Middle Miocene, ca. 23-15 Ma. [24][25] [29][30][31][32]. Another outcrop near Simojovel assigned to the La Quinta strata dated as late Oligocene has been mentioned as containing amber [29] [30]. However, the location of that outcrop cannot be confirmed in current field studies [24] [25]. Other author also discuss the stratigraphic position of a section of the Los Pocitos site near Simojovel-this locality also contains amber lumpsby placing it in the Simojovel Formation, which informally correlates with the La Quinta Formation in the Late Oligocene [33]. However, this age correlation is based on unpublished, unavailable biostratigraphic report apparently using Foraminifera as mentioned very briefly in [33], which increased confusion about the amber age. Geology of amber outcrops in the Mountains of Chiapas, from Totolapa to Estrella de Belén near Palenque, should be correlated with more supporting stratigraphic data in further published works.
The Guadalupe Victoria site near Simojovel along with other amber sites grouped in the Mountains of Chiapas constitute a Miocene Conservation Lagerstätte with paleobiota conspicuously preserved [24] [25]. Those fossils show hard and soft tissues preservation in remarkable detail. The preservation potential has been influenced by rapid resin hardening, followed by a restriction of chemical reactions and incomplete organic decay [34]. Since the initial work by Langenheim (1966), the plant source of Chiapas amber is assigned to an extinct legume tree species of the genus Hymenaea, because it shares chemical signatures with extant resins of the living tree species H. courbaril and H. verrucosa [35][36][37]. The recent biogeochemical features and organic mineral nomenclature of Chiapas amber has also been recently reviewed [38].
The Chiapas amber also shares botanical affinities with those fossil resins occurring in the Caribbean since the Miocene, such as amber from the Dominican Republic, Haiti, Puerto Rico, Cuba, and Jamaica [30][36] [39]. Recently, this amber group is known as the Middle America amber in the Neogene [24] [25]. These amber strata also have the regional tectonic setting (in the Caribbean Plate and the Central America formation) and a sedimentary regimen in common with the Simojovel sites at the Miocene [40]. However, the age correlation of amber deposits exposed in Chiapas and the Caribbean group should be interpreted with caution, because there are significant differences in depositional ages (still unstudied) that should be considered at detail.

Material and Methods
The amber scorpion was collected in hand-made pit near the Guadalupe Victoria site, Latitude 17°09´11´´N, Longitude 92°46´08´´W, located near Simojovel, Chiapas, southern Mexico (Fig 1). The specimen is now housed at the Museo del Ámbar de Chiapas (MACH) located in San Cristóbal de Las Casas, Chiapas, Mexico. The amber collection of the MACH, including the fossil scorpion, is formally certified by the Instituto Nacional de Antropología e Historia (INAH), which protects the archeological and paleontological heritage in Mexico. No specific permits were required for the specimen description and paleontological fieldwork, which complied with all relevant regulations.

Micrographs and measurements
Anatomical data were collected using a high-resolution microscopy with regular to adapted infrared-pass lens with LED/tungsten lamps as infrared source. A multiple image superimposition with >16 planes per image were applied for images processing and displaying [24][25] [34]. Schematic drawings were hand traced using an electronic pen and a stereomicroscope, micrographs and CorelDraw X6 for graphic processing. Anatomical measurements (given in millimeters) were collected using the open source program tpsDig V. 2.17 [41]. Provenance analysis on amber was applied using infrared and X-ray spectroscopies [34] [38].

Phylogenetic analysis
The phylogenetic position of Tityus apozonalli sp. nov. among its fossils congeners from both Dominican and Mexican amber, Neogene, was analyzed using the TNT program [42]. Branch support values were calculated with the function tree bisection and reconnection (TBR) from existing trees, and character mapping and bootstrap values on the most parsimonious tree using a traditional heuristic search with 100,000 replicates. S1 Table shows the data matrix that includes the most informative morphological characters from the following fossil taxa: T. (Brazilotityus) hartkorni [20], T. geratus [19], T. azari [21], T. (Brazilotityus) knodeli [3], C. beynai [13], M. ambarensis [16] [17] [18], and T. apozonalli. Palaeoananteris ukrainensis from Rovno amber, Paleogene [11], was used as the outgroup because of its relatively basal condition and comparable plesiomorphic characters. Distribution of qualitative and quantitative characters among taxa has been listed in S1 Appendix.

Nomenclature
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code in the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lisd:zoobank.org:pub:65DAA8C5-63DF-4995-BABA-992395A9747B. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS

Derivation of name
"apozonalli", from the Náhuatl language. This term was given to amber in the pre-Columbian times by the Aztecs, and literally means "sea bubble" or "sea foam".

Type material
BLMACH.18, amber inclusion, three-dimensionally preserved, entire adult male and only specimen known (Figs 2 and 3). Currently is housed at the Museo del Ámbar de Chiapas (MACH), located in San Cristóbal de Las Casas, Chiapas, Mexico.

Locality and Horizon
The Guadalupe Victoria site, latitude 17°07´58´´N, longitude 92°48´19´´W, located in the Municipality of the Simojovel de Allende, Chiapas, Mexico (Fig 1). The amber-bearing beds are part of the Mazantic shale and Balumtum sandstone strata from the Early-Middle Miocene (23-15 Ma).

Taphonomic features
The specimen is embedded in golden yellow amber, tinged with orange, showing cloudy to weakly translucent glossiness, with abundant oblique microfractures (Fig 2). There is a recrystallization pattern (wave-shaped) close to the body, which is a consequence of different crystallization stages during resin hardening (Fig 2) Both fractures and recrystallization have darkened some minute anatomical elements, i.e. trichobothria, carinae and macrosculpture, which are hard to distinguish (Fig 3). There is soil, plant remains, insect parts, bubble molds and undetermined minerals embedded together with the scorpion (Fig 2). Most portions of the carapace and tergites are compressed, almost obliterated, and somewhat translucent, as a consequence of organic decay (Fig 2). This suggests that organic acids in amber have partially dissolved the cuticle. Amber is still chemically reactive in the depositional environment; this affects the organic preservation at different levels [33]. However, pedipalps, chelicerae, caudal segments and telson are less dissolved, staying tightly sclerotized (Figs 3, 5 and 7). Undetermined mineral salts are spread under sternites. These are secondary reactive products from the resin hardening. Pectines and adjacent sternites are slightly bleached (Fig 5). Legs are also translucent, flattened, with inner soft-tissues heavily degraded (Fig 7A). Telson is basally broken close to anus. The true color morphology pattern is well-preserved in general (Fig 7B and 7C).
Tityus is a highly diverse group whose genus-level taxonomy and evolutionary relationships is currently problematic. In the past two decades, there has been important progress in identifying and separating related subclades within Tityus based on morphological characters, such as the subgenera proposed by Lourenço (2006) [23]. An attempt to resolve the phylogenetic relationships of Tityus is beyond the scope of the present paper. However, regarding the phylogenetic status of T. apozonalli among its fossil buthid congeners from both Dominican and Mexican amber, which are preliminary examined herein, the results show a single most parsimonious tree with length (L) = 27, consistency index (CI) = 0.77 and retention index (RI) = 0.72 (Fig 9). The resulting cladogram indicates a basal position of T. apozonalli which is separated from T. azari and T. (Brazilotityus) hartkorni from Dominican amber, as was T. (Brazilotityus) knodeli, previously described in Chiapas amber (Fig 9). T. apozonalli shows a plesiomorphic condition instead of T. (Brazilotityus) knodeli that seems more derived and closer to C. beynai and M. ambarensis. The plesiomorphic condition in T. apozonalli is supported consistently, but not exclusively, by the discernible fulcra that are also present in the 'Tityus clathratus' morphotypes, including initially within the Tityus (Archaeotityus) subgenera [23].
As shown in the resulting tree, the fossil members of Tityus seems paraphyletic, the grade of paraphyly is resolved at low resolution because of living species are not included in this analysis. However, this phylogenetic analysis provides first insights into previously unclear relationships between Tityus fossil members, supporting that T. apozonalli has a basal condition. Paraphyly is observed in fossil taxa with transitional states [50]. It has been discussed that fossil species with a paraphyletic behavior probably emerged by populations branching off from an ancestor who was not subsequently extinguished or as the result of adaptation by spatial differentiation, also paraphyly in fossil species might represent an extinction event [50]. Thus, the fossil diversity of Tityus as observed in a given time (Miocene) and space (Middle America) is discontinuous and might suggest extinction clues (Fig 9). Further phylogenetic analyses by using morphological and molecular data from living species must necessarily clarify many unknown relationships in this hugely diverse group.
The extant forms of the 'Tityus clathratus' subclade close to T. apozonalli currently live in the Neotropics of Central and northern South America [51], whereas the distribution of T. apozonalli is limited to the Chiapas amber in the southernmost part of North America during the Miocene. The current distribution of the living buthid scorpions is worldwide except Antarctica [6]. As shown by the fossil record, the family Buthidae is represented by modern forms that occur since the Paleogene of Europe with several genera found in Baltic amber, as well as single genus Uintascorpio found in the Paleogene of North America [14][15] (Fig 10). Several published contributions suggest that extant buthid scorpions from Asia, Africa and South America have a Gondwanaland relationship [6]. Although there is a notable gap in the Mesozoic fossil record (Fig 10), it has been preliminarily considered that the buthid scorpions emerged in Gondwana (near the Permian-Triassic boundary) as predicted by the current worldwide distribution [6]. The superfamily Buthoidea with two families: Buthidae and Microcharmidae, is considered monophyletic, neither the family Archaeobuthidae nor Palaeoburmesebuthus (family Incertae sedis) are currently included in the Buthoidea [6]. The assignment of the Triassic family Protobuthidae into Buthoidea is still inconclusive (Fig 10). Also, the placement of the family Microcharmidae Lourenço, 1996 as separate from the Buthidae is still under debate [6] [52]. Based on mesosomal anatomy other authors propose a synonymy of Microcharmidae = Buthidae [53].
On the other hand, the genus Tityus is typically a New World group. The fossil specimen 'Tityus' eogenus from the Baltic amber (Central to North of Europe) seems to be initially misplaced [12]. Currently the type material of 'Tityus' eogenus is missing [12]. There are also three buthid species found in the Recent Madagascar copal (less than 250 years old); one species was  [52] [55]. However, these may be considered as extant species, not belonging to the fossil or even subfossil record due to the recent depositional age of copal (Fig 10).

Conclusion
Tityus apozonalli sp. nov. brings the number of scorpions known from the Chiapas amber to three. However, the fossil record also includes at present other four unidentified specimenslikely Tityus morphotypes-now kept in some private collections and several missing specimens as result of amber fossil trading. Historically, invaluable Chiapas amber fossils have been accumulated in anachronistic and pretentious collections that illegally export fossils from Mexico. Unfortunately, the type material of T. (Brazilotityus) knodeli and Centruroideslike specimen that were mentioned earlier fall into this ambiguous context [3] [22]. Thus, the specimens revision for comparison is basically prohibited and the information unverifiable. Also the provenance of amber pieces is questionable because Mexican amber is roughly equal to Dominican, i.e. in color, glossiness, general composition, associated plant source, similar paleobiota and geological age [34][35][36][37][38]. Accordingly, T. apozonalli is the first fossil scorpion in the Chiapas amber with unquestionable repository and provenance, which is particularly relevant for further research.
Endemism and species richness of the scorpion fauna in Mexico is conspicuously high compared to worldwide, with 258 described species, assigned to 26 genera and 7 families, including Buthidae, Chactidae, Diplocentridae, Euscorpiidae, Iuridae, Superstitioniidae, Typhlochactidae and Vaejovidae, which are distributed in both Nearctic and Neotropical environments [56] [57]. Currently, the average number of known species from Mexico is nearly 13.5% of the worldwide diversity [57], but the number is growing regularly as new species are described and taxonomic categories re-evaluated. The family Buthidae in Mexico comprises different extant species of the genus Centruroides, which is widely distributed in the territory [57][58], another extant species from Oaxaca near Chiapas [59] that was initially assigned to the genus Tityopsis Armas, 1974 but recently transferred to a new genus Chaneke Francke, Teruel and Santibañez-López 2014 [60], and two fossil species of Tityus from the Chiapas amber that now includes T. apozonalli [3]. The fossil Centruroides-like morphotype reported earlier needs revision [22].
Notably, there are no living species of the genus Tityus which have been previously described in Mexico. Tityus essentially shows a Neotropical distribution from northern South America to Central America and the Caribbean Islands [23]. Fossil species of Tityus from Chiapas amber now extend the geographical range of the genus to the southernmost part of North America in the Miocene. According to Lourenço (2014), primitive Tityus were likely replaced by opportunistic Centruroides which now are prevalent in southern Mexico [3]. However, the sedimentary and tectonic evolution of southern Mexico must play an important role in the extinction and dispersal of the Chiapas amber paleobiota, including the scorpion fauna, since the beginning of the Miocene to mid-Pliocene, ca. 23-5 Ma [61]. Accordingly, the extinction and consequent dispersion to new endemic areas was probably induced by land changes [61]. Here a vicariance interpretation suggests that past geological events in Chiapas have changed the range of amber paleobiota. The current distribution of Tityus scorpions supports this hypothesis.
The biogeographic relationships between multiple species of Tityus distributed in the northern South America and the Caribbean Islands are pointed out in several contributions [23][62] [63]. As noted, the presence of discernible fulcra in T. apozonalli from Chiapas as shown in other small-sized fossil Tityus morphotypes from the Middle America amber seems to be a relict that shared with the present-day specimens of the Tityus (Archaeotityus) group, which now occur living extensively from northern South America to the Caribbean Islands. This also suggests an intense radiation at least since the Neogene in the Neotropics of southernmost North America.
Supporting Information S1 Appendix. List of characters scored for phylogenetic analysis. (DOCX) S1