New record of Shiny Peeping Frog, Eleutherodactylus nitidus (Peters, 1870) (Anura, Eleutherodactylidae), in Mexico City, Mexico

. Based on molecular and morphological evidence, we report the first record of Eleutherodactylus nitidus (Peters, 1870) from Mexico City, in the urban area of Panteón Xilotepec, Xochimilco. We discuss the remarkable urban adaptability of Eleutherodactylus and the need for additional sampling to determine if E. nitidus is native or introduced to Mexico City.

Eleutherodactylus (Syrrhophus) nitidus (Peters, 1870) is endemic to Mexico, where it has been recorded in the states of Guerrero, Oaxaca, Mexico, Morelos, Tlaxcala, and Puebla (Grünwald et al. 2021;Devitt et al. 2023).While this species has not been previously reported in Mexico City, Lemos-Espinal and Smith (2020) suggested the possibility of its occurrence there due to its distribution in the nearby states of Mexico and Morelos, in particular in the southeast, where a large conservation area covers approximately 59% of the city (Reygadas-Prado 2016).
Here, we use genetic and morphological evidence to verify and confirm an E. nitidus specimen from Mexico City, establishing the first published occurrence of this species in this highly urbanized region.

METHODS
In July 2023 we heard Eleutherodactylus calls in the cemetery of Panteón Xilotepec, which is located in Xochimilco, Mexico City.We later collected a specimen on the wall in the cemetery under permit issued to Uri Omar García-Vázquez by the Mexican goverment's Secretaría de Medio Ambiente y Recursos Naturales (Faut-0246).We euthanized the specimen using 20% benzocaine gel, preserved a liver sample in absolute molecular-grade alcohol, and fixed the specimen in 10% formalin (Beaupre et al. 2004).This specimen is deposited in the Herpetological Collection of the Museo de Zoología, Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México (MZFZ).To verify its taxonomic identity, we compared this specimen with the original descriptions of E. nitidus (Peters, 1870), E. grandis (Dixon, 1957), and E. maurus (Hedges, 1989).These are the three species of Eleutherodactylus, subgenus Syrrhophus, with the closest distribution to Mexico City.Our morphological examination focused on the measurements used by Peters (1870): snout to vent length (SVL), head width (HW), diameter of tympanum (TD), diameter of eye (EL), tibia length (TL), and hand length (HAL), averaged with an electronic vernier callipers.In addition, distinctive phenotypic characteristics of the species mentioned by Devitt et al. (2023) and Grünwald et al. (2023) were identified such as: dorsal skin texture, form of canthus rostralis, form of snout, and color pattern.
Additionally, we extracted DNA from the liver sample using a DNeasy Blood & Tissue DNA extraction kit (Qiagen Inc., Valencia, CA, USA).We verified DNA quality by electrophoresis using a 1kb Plus molecular weight marker (100-12,000 bp) and visualized it in an ultraviolet light chamber (UV-Transilluminator).From the extracted DNA, we amplified a fragment of the 16S ribosomal gene through polymerase chain reaction with primers 16Sar (Palumbi 1996) and 16H47 (Heinicke et al. 2007).We purified PCR products using polyethylene glycol (Lis 1980).Finally, the purified sample was sequenced by Macrogen Inc. in South Korea.
To determine its phylogenetic position, we obtained from GenBank 83 sequences of the 16S rRNA gene from the subgenus Syrrhophus, including species related to E. nitidus, such as E. grandis, and E. maurus; additionally, we used a sequence of Eleutherodactylus zeus Schwartz, 1958 as an outgroup (Appendix, Table A1) (Devitt et al. 2023;Grünwald et al. 2023).We edited the sequence of our new specimen in Sequencher v. 4.1.4(Gene Code Corporation, USA), and along with the GenBank sequences we aligned it in UGENE 47 (Okonechnikov et al. 2023) using the MUSCLE algorithm (Robert 2004).Subsequently, we used PartitionFinder v. 2 (Lanfear et al. 2017) to choose the partitioning scheme under the greedy search algorithm using a Bayesian Information Criterion.Finally, we constructed a phylogenetic tree in RAxML v. 8.2.X software (Stamatakis 2014) with the graphical interface raxmlGUI v. 2.0 (Edler 2021) using 1000 ultrafast bootstrap replicates to assess branch support.tympanum barely visible through the skin, (5) mottled lip margins, and (6) brown-banded limbs (Figure 1).In life, the dorsal coloration is green, with a brownish tone due to skin granules.The green coloration is more intense on the extremities, which also have brown bands.The lumbar gland is white with black spots that extend laterally on both sides of the body.The venter is pale, as are the legs (Figure 1).In comparison to E. grandis, our new specimen is smaller in size (E.grandis: SVL = 30.5 mm), with a narrower head (E.grandis: HW = 11.7 mm), and a smaller tympanum (E.grandis: TD = 2 mm).The latter characteristic of the specimen collected is shared with E. maurus (a small, inconspicuous tympanum), but unlike the latter it lacks a black line anterior and posterior to the eye (present in E. maurus), has a mostly white lumbar gland (mostly black in E. maurus), and has pale lips with greenish spots (dark lips with pale speckles in E. maurus) (Peters 1870;Davis and Dixon 1955;Dixon 1957;Devitt et al. 2023;Grünwald et al. 2023).

Molecular analysis.
We included 84 sequences in our phylogenetic analysis, and in the final alignment we obtained a matrix of 598 base pairs for the 16S rRNA gene across 32 species of the genus.
The new specimen collected in Mexico City (MZFZ 4580) grouped within the E. nitidus clade and is sister to the genetic samples from the geographically proximate State of Mexico and Morelos (bs = 63) (Figure 2).

Additional records.
At the site where we collected the specimen, other Eleutherodactylus calls were heard, indicating the presence of more individuals (Supplemental data, Audio File S1).

DISCUSSION
The origin of Eleutherodactylus nitidus in Mexico City is uncertain.The examined records might be native due to their proximity and phylogenetic affinity to records from the State of Mexico and Morelos.The distance between our new record and closest previously known occurrence in Morelos, which bords the southern part of Mexico City, is 27 km.Here, in this part of the city, Lemos-Espinal and Smith (2020) speculated that E. nitidus might be present in forests and ravines.However, human transport cannot be ruled out, because Kraus et al. (1999) and Kaiser et al. (2002) mentioned that Eleutherodactylus species have been introduced through the trade of ornamental plants.
Although most specimens are collected from wildland areas, urban parks are increasingly recognized as conducive to maintaining Syrrhophus populations.The documented adaptability of E. nitidus to urban settings has been underscored in prior studies.This adaptability has been attributed to these frogs' small size and to their direct developing life history, which frees them from requiring large areas of habitat or standing water to survive and reproduce (García-Vázquez and Trujano-Ortega 2012; Gómez-Benitez et al. 2021; Vázquez-Hernández 2023).Our new record from Mexico City adds to this steadily growing documentation of E. nitidus from highly urbanized areas.
In the absence of definitive historical data to classify species as native or non-native to an area, phylogeographic and genetic analyses are valuable tools.These analyses can reveal genetic signatures of the founder effect for introduced populations, which often have reduced genetic variation due to rapid population expansion, have more recent divergence dates, and are less likely to be genetically structured compared to populations from their native range (Yuan et al. 2022).Records of the genus in the city may be misidentified as E. grandis, given the restricted distribution of the species (Serrano-Serrano, 2016).To know how many species of the genus are currently in the city and to decide if E. nitidus is native or non-native, an extensive sampling is necessary across a larger geographic area.

Figure 2 .
Figure 2. Phylogenetic relationships of the 16S gene obtained from the maximum-likelihood analysis of the members of Eleutherodactylus subgenus Syrrhophus.Numbers in the nodes represent the bootstrap support value.All nodes at species level were collapsed except for E. nitidus.