Use the following type of citation: North-western Journal of Zoology 2021: e217505
Paper Submitted to The North-Western Journal of Zoology
1 *Handling editor: Yurii Kornilev
2 *Manuscript Domain: Herpetology of the Americas
3 *Manuscript code: NwJZ_21_HA_04
4 *Submission date: 02_03_2021
5 *Revised: 09_06_2021
6 *Accepted: 05_08_2021
7 *No. of words (without abstract, acknowledgement, references, tables, captions): 1330 8 (papers under 700 words are not accepted)
9 *Editors only:
10 11 Title of the paper: Molecular identification of the Jalisco Trilling Frog Eleutherodactylus
12 jaliscoensis (Amphibia: Eleutherodactylidae) as prey of theZoology Garter Snake Thamnophis of 13 cyrtopsis (Squamata: Colubridae) proofing 14 Journal 15 Running head: Eleutherodactylus jaliscoensis as preyuntil of Thamnophis cyrtopsis
16 paper 17 Authors (First LAST - without institution name!): Raquel HERNÁNDEZ-AUSTRIA, Ángel Fernando
18 SOTO-POZOS, Omar BECERRA-SORIA, Abigail MORA-REYES, Gabriela PARRA-
19 OLEA North-western Accepted 20
21 Key Words (at least five keywords): Mexico, endemism, relationship predator-prey, diet,
22 mtDNA16S.
23
24 No. of Tables: 0
25 No. of Figures: 3
26 No. of Files (landscape tables should be in separate file): 1 Use the following type of citation: North-western Journal of Zoology 2021: e217505
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27 Molecular identification of the Jalisco Trilling Frog Eleutherodactylus jaliscoensis
28 (Amphibia: Eleutherodactylidae) as prey of the Garter Snake Thamnophis cyrtopsis
29 (Squamata: Colubridae)
30 Raquel HERNÁNDEZ-AUSTRIA1,2, Ángel Fernando SOTO-POZOS1,2, Omar BECERRA-
31 SORIA1,2, Abigail MORA-REYES3 and Gabriela PARRA-OLEA1,*
32
33 1. Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de
34 México, A.P. 70-153, Ciudad Universitaria, C.P. 04510, Mexico City, Mexico.
35 2. Posgrado en Ciencias Biológicas, Unidad de Posgrado, Edificio A, 1° Piso, Circuito de
36 Posgrados, Ciudad Universitaria, Coyoacán, C.P. 04510, Mexico City, Mexico. Zoology 37 3. Universidad Veracruzana, Facultad de Ciencias Biológicas y Agropecuarias, Campus of 38 Peñuela, camino Peñuela-Amatlán, S/N Peñuela Amatlán de los Reyes, C.P. 94945, Veracruz, proofing 39 Mexico. Journal 40 until
41 *Corresponding author, Gabriela Parra-Olea, E-mail: [email protected] paper 42
43 Abstract. We report the predation of the recently described and poorly know frog
44 Eleutherodactylus jaliscoensisNorth-western by the snake Thamnophis cyrtopsis employing a mitochondrial Accepted 45 marker molecular and morphological evidence. We discuss the importance of frogs in food
46 webs and the use of sequences de DNA in the identification of frogs.
47
48 Key Words: Mexico, endemism, predator-prey relationship, diet, mtDNA16S.
49 Running title: Eleutherodactylus jaliscoensis as prey of Thamnophis cyrtopsis
50 Use the following type of citation: North-western Journal of Zoology 2021: e217505
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51 Direct developing frogs of genus Eleutherodactylus are one of the most diverse groups of
52 vertebrates in the Neotropics with more than 200 species described (Lynch & Duellman 1997,
53 AmphibiaWeb 2021, Frost 2021), which are distributed from the southern USA to northern
54 Argentina (Padial et al. 2014). In Mexico, there are 37 described species of Eleutherodactylus
55 classified in the subgenus Syrrhophus (Hedges et al. 2008, Grünwald et al. 2021), with most
56 of them having a restricted distribution.
57 The Jalisco Trilling Frog Eleutherodactylus (Syrrhophus) jaliscoensis is an endemic species
58 to the state of Jalisco, Mexico. This species is distributed in pine-oak forests in the Sierra
59 Mascota, Sierra Cuale, and Sierra Cacoma within the Transmexican Volcanic Belt
60 biogeographic province, ranging from 1,700 to 2,300 m a.s.l. (Grünwald et al. 2018). This Zoology 61 species has been collected at ground level among oaks, pines, and herbaceous plants of 62 (Grünwald et al. 2018). Eleutherodactylus jaliscoensis is an endangered species according to proofing 63 the IUCN Red List of Threatened Species (2021); but it is not included in the list of species at Journal 64 risk of the Norma Oficial Mexicana NOM-059-SEMARNAT-2010.until Being a recently
65 described species, little is known about the biotic interactions and its natural history, as well paper 66 as its predators and prey, and other aspects of basic biology.
67 The distribution of E. jaliscoensis is immersed in the distribution of the Black-necked Garter
68 Snake Thamnophis cyrtopsisNorth-western, a widely distributed snake from the southern United States, Accepted 69 through Mexico to Guatemala, in vast types of environments ranging from tropical,
70 temperate, to arid climates, and elevations from sea level to 3,000 m a.s.l. (Wilson & Johnson
71 2010). In the highlands, T. cyrtopsis prefers mountain streams but in summer rains sometimes
72 it is found some distances from permanent water (Stebbins 1954). This species is considered
73 an aquatic-foraging generalist and feeds on a wide variety of prey, including semi-aquatic and
74 terrestrial organisms (Jones 1990). Amphibian prey reported for T. cyrtopsis included adult
75 and larvae of Lithobates catesbeianus, possibly some larvae of L. pipiens (Fleharty 1967), Use the following type of citation: North-western Journal of Zoology 2021: e217505
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76 adult of L. chiricahuensis, L. yavapaiensis, and Dryophytes arenicolor (Jones 1990, McCall et
77 al. 2017).
78 Here we report both the first record of consumption of E. jaliscoensis by the snake T.
79 cyrtopsis, and in turn, some prey items consumed by this species of frog. Our observation
80 provides an important addition to the natural history and knowledge of predator-prey
81 relationships of E. jaliscoensis and T. cyrtopsis.
82 During a nocturnal field survey in August 2019 to collect individuals of E. jaliscoensis in the
83 type locality (Sierra Mascota, 20°41'51.52” N, 104°52'0.72” W, datum WGS84, 2,083 m
84 a.s.l.; Fig. 1), Jalisco, Mexico, we collected a E. jaliscoensis (IBH32593) and a T. cyrtopsis
85 (IBH32595). The E. jaliscoensis was found on the forest floor devoid of bushes, while T. Zoology 86 cyrtopsis was found under a rock amongst many bushes around. We obtained liver tissue from of 87 each specimen and stored in RNAlaterTM Tissue Storage Reagent. The specimens were proofing 88 killed with an overdose of sodium pentobarbital, fixed in 10% formalin solution, and Journal 89 preserved in 70% ethanol and deposited in the Colecciónuntil Nacional de Anfibios y Reptiles
90 (CNAR), Instituto de Biología, Universidad Nacional Autónoma de México. paper 91 We found the remains of a frog in the T. cyrtopsis' stomach (Fig. 2A). The posterior region of
92 the frog was in good condition; therefore, we obtained a sample of muscle tissue from the
93 thigh, which we molecularlyNorth-western analyzed for taxonomic identification. Additionally, we Accepted 94 reviewed the remaining parts using a Carl Zeiss (model 2004017913) dissecting microscope.
95 We extracted DNA from the thigh tissue using phenol-chloroform protocol (Sambrook &
96 Russell 2006) and sequenced a region of the 16S mitochondrial gene with the primers 16Sar
97 and 16Sbr (Palumbi et al. 1991). We aligned the new sequence with sequences of 18
98 Eleutherodactylus species including the sequence of the E. jaliscoensis type specimen
99 (accession number MG857003) from the GenBank online repository, using the MAFFT v.7
100 online (Katoh et al. 2019). We obtained the best nucleotide substitution models with Use the following type of citation: North-western Journal of Zoology 2021: e217505
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101 PartitionFinder v.1.1 (Lanfear et al. 2012), resulting in the GTR + I + G as the best. We
102 performed a Bayesian inference analysis in the MrBayes 3.2.3 program (Ronquist et al. 2012),
103 with two independents runs of 10,000 million generations each that were sample every 1,000
104 generations. We discarded the first 25% (2,500,000) of trees as burn-in and used the
105 remaining trees to estimate the majority consensus tree. We made the analysis on the Cipres
106 Science Gateway (Miller et al. 2010). We visualized and edited the generated tree in FigTree
107 v.1.4.3 (Rambaut 2007). Additionally, we used the Basic Local Alignment Search Tool
108 (BLAST) to compare the generated sequence against other Eleutherodactylus sequences. The
109 sequence obtained was deposited in the GenBank repository (accession number MW605049).
110 The Bayesian phylogenetic tree grouped the new sequence with a series of sequences of E. Zoology 111 jaliscoensis; this clade is supported by a high nodal value in our analysis (Pp = 0.95) (Fig. 3). of 112 Moreover, the 16S gene sequence obtained, matched with E. jaliscoensis sequences with a proofing 113 99.64% identity and an e-value of 3e-139. Our results confirm that the specimen (IBH32594) Journal 114 found in the stomach of T. cyrtopsis belongs to E. jaliscoensisuntil . Additionally, some phenotypic
115 characteristics that were observed in the undigested posterior region of the specimen paper 116 (crossbars present in the thighs and legs, and the inner surface of the thigh red) (Fig. 2A, B)
117 coincide with diagnostic characters reported by Grünwald et al. (2018) for E. jaliscoensis.
118 Furthermore, we wereNorth-western able to rescue and identify some prey in the E. jaliscoensis' stomach. Accepted 119 All preys were arthropods of the orders Araneae (family Anyphaenidae), Acari (family
120 Oribatellidae), and Coleoptera, the latter both in its larval (not identified family) and adult
121 stages (including the family Elateridae and another not identified); and remains of
122 unidentified material (Fig. 2C).
123 The encounter of a second individual of E. jaliscoensis but in T. cyrtopsis' stomach was quite
124 unexpected. The two specimens reported in this study (IBH32593 and IBH32594) plus three
125 specimens reported by Grünwald et al. (2018) represent a total of five specimens of E. Use the following type of citation: North-western Journal of Zoology 2021: e217505
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126 jaliscoensis collected at the type locality, which suggests this species may not be abundant in
127 the Sierra Mascota. More research about population size, life history, distribution, genetic
128 diversity, and threats are needed to help us to better understand and conserve this species.
129 Also, our findings display the role of frogs as an important component of the trophic
130 networks, both as part of the diet of other vertebrates as generalist predators and major
131 consumers of arthropods, emphasizing their role as biological controllers of invertebrate
132 populations.
133 That same night, we recorded three individuals of E. teretistes and the snakes Conopsis nasus,
134 Leptodeira splendida, and Geophis bicolor. In this sense, the presence of both prey and
135 predators in the environment (in this case, frogs, and snakes) could be a key bioindicator of Zoology 136 environmental health. The absence of predators would be an indicator that their food is of 137 decreasing too. Our findings indicate that Sierra Mascota holds a diverse fauna, however, it proofing 138 does not belong to any protected natural area. Furthermore, the recent description of E. Journal 139 jaliscoensis from the Sierra Mascota (Grünwald et untilal. 2018) suggests that this site has not yet
140 been fully explored. paper 141 We stress the importance of generating information about the biology of species, especially
142 those reduced distributions and endemic species, that for different reasons (e.g. recent
143 discovery or lack ofNorth-western fieldwork) are not contemplated in any national or international Accepted 144 conservation status system. The more biological information on the species we have, the more
145 accurate evaluations regarding their threat or conservation will be and this information is
146 important to make effective conservation strategies.
147 Finally, we show the usefulness of the 16S mitochondrial marker as a powerful tool for the
148 identification of amphibian species, as demonstrated in other studies (Grünwald et al. 2021,
149 Freitas et al. 2021), even when the tissue sample presents a degree of deterioration. In this
150 case, it was possible to obtain enough DNA from muscle tissue of a frog that was semi- Use the following type of citation: North-western Journal of Zoology 2021: e217505
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151 digested to be used in other biological studies, for example, of molecular phylogenetics and
152 population genetics.
153 Acknowledgments
154 This research was supported by UNAM PAPIIT (Project # 205521) to GPO. We thank
155 Posgrado en Ciencias Biológicas of the Universidad Nacional Autónoma de México (UNAM)
156 for its support to this study; Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico)
157 for providing scholarships to RHA (CVU 662067), AFSP (CVU 742781), and OBS (CVU
158 328535); Secretaría del Medio Ambiente y Recursos Naturales in Mexico for Collection
159 Permit SGPA/DGVS/003513/18; J. D. Lara-Tufiño for his help in the identification of T.
160 cyrtopsis and comments on drafts of this manuscript; D. Guerrero and N. Gutiérrez for their Zoology 161 help in the identification of arthropods. We are grateful to C. I. Grünwald, an anonymous of 162 reviewer, and the handling editor Y. Kornilev for helpful comments in improving the proofing 163 manuscript. Journal 164 until
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201 comments on the taxonomy of related species and their advertisement calls.
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225 Wilson, L.D., Johnson, J.D. (2010): Distributional patterns of the herpetofauna of
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228 Eagle Mountain Publishing.
229
230 Table captions (if applicable)
231
232 Figure captions (if applicable)
233
234 Figure 1. Geographic location (red circle) of the collected individuals of T. cyrtopsis Zoology 235 (IBH32595) and E. jaliscoensis (IBH32593 and IBH32594) in the Sierra Mascota, Jalisco, of 236 Mexico. proofing 237 Journal 238 Figure 2. (A) Thamnophis cyrtopsis with an individualuntil of E. jaliscoensis in its stomach; (B)
239 Dorsal view of body portion of E. jaliscoensis with crossbars on thighs and legs; (C) Ventral paper 240 view of body portion with red colour on the inner thighs; (D) Microscopic images of prey
241 types found in E. jaliscoensis' stomach (photo not to scale): (D1) One spider (Araneae); (D2)
242 Two mites (Acari); (D3)North-western Larva and (D4) adult of beetle (Coleoptera), the upward arrow Accepted 243 indicates four incomplete beetles (heads and elytra), and the downward arrow indicates a
244 complete beetle. The remnant material represents unidentified material.
245
246 Figure 3. Phylogenetic position of the DNA sample of E. jaliscoensis from the Sierra
247 Mascota, Jalisco based on sequences of the 16S mitochondrial gene. In bold, the sequence
248 generated in this study (MW605049) and the sequence of the E. jaliscoensis type specimen
249 from GenBank (MG857003). Asterisks indicate Posterior probabilities ≥ 0.95. Use the following type of citation: North-western Journal of Zoology 2021: e217505
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