Published in the United States of America 2024 * VOLUME 18 * NUMBER 1 & 2
AMPHIBIAN & REPTILE
CONSERVATION
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ISSN: 1083-446X eISSN: 1525-9153
Amphibian & Reptile Conservation 18(1&2) [General Section]: 1-9 (e329).
Official journal website: amphibian-reptile-conservation.org
Conservation status, range extension, and call analysis of the Littoral Glassfrog, Cochranella litoralis (Ruiz-Carranza and Lynch 1996)
"Ross J. Maynard, José Tinajero-Romero, *Sebastian Kohn, *Christophe Pellet, and **Jaime Culebras
'The Biodiversity Group, Tucson, Arizona, USA Pontificia Universidad Catélica del Ecuador, Museo de Zoologia, Quito, ECUADOR *Fundacién Condor Andino, Quito, ECUADOR “Bosque Protegido “El Jardin de los Suetios,” Los Laureles, La Mand, Cotopaxi, ECUADOR *Photo Wildlife Tours, Quito, ECUADOR
Abstract.—The little-known glassfrog Cochranella litoralis (Ruiz-Carranza and Lynch 1996) is a Vulnerable (VU) species infrequently reported in the literature. Its purported distribution includes the departments of Cauca and Narino, Colombia, and the provinces of Esmeraldas, Los Rios, Pichincha, and Santo Domingo de los Tsachilas, Ecuador. Due to conflicting details regarding its distribution within the literature, we review past records to clarify which localities are valid. We also report two new localities that expand its elevational range to < 407 m and its distribution approximately 175 km south from the previous southernmost locality, present an updated distribution map, and recommend an IUCN Red List status of Endangered (EN) for C. /itoralis. Lastly, the call of C. litoralis is described for the first time, as is that of an Ecuadorian specimen of the widely-distributed C. granulosa.
Keywords. Amphibian, Anura, distribution, Ecuador, Endangered, threatened
Citation: Maynard RJ, Tinajero-Romero J, Kohn S, Pellet C, Culebras J. 2024. Conservation status, range extension, and call analysis of the Littoral Glassfrog, Cochranella litoralis (Ruiz-Carranza and Lynch 1996). Amphibian & Reptile Conservation 18(1&2): 1-9 (e329).
Copyright: © Copyright: Maynard, et al. 2024. This is an open access article distributed under the terms of the Creative Commons Attribution License [Attribution 4.0 International (CC BY 4.0): https://creativecommons.org/licenses/by/4.0/], which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The official and authorized publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation; official journal website: amphibian-reptile-conservation.org.
Accepted: 13 February 2024; Published: 1 June 2024
Resumen.—La poco conocida rana de cristal Cochranella litoralis (Ruiz-Carranza y Lynch 1996) es una especie Vulnerable (VU) con pocos registros en la literatura. Su distribucion conocida incluye los departamentos de Cauca y Narino, en Colombia, y las provincias de Esmeraldas, Los Rios, Pichincha y Santo Domingo de los Tsachilas, en Ecuador. Debido a que varias fuentes tienen detalles contradictorios con respecto a su distribucion, revisamos los registros para mayor claridad, reportamos dos nuevas localidades que amplian su rango de altitud a < 407 my su distribucion aproximadamente 175 km al sur de la localidad mas al sur conocida, presentamos un mapa de distribucion actualizado, y recomendamos que el estado de la Lista Roja de la UICN de C. litoralis se modifique a En peligro (EN). Por ultimo, se describe por primera vez la llamada de C. /itoralis, asi como la de un ejemplar ecuatoriano de la ampliamente distribuida C. granulosa.
Palabras Claves. Anfibio, Anura, distribucion, En peligro, amenazada
Introduction
The glassfrog genus Cochranella was first proposed over 70 years ago and included 13 species at that time (Taylor 1951). More recently, the genus was revised to resolve its former polyphyly, which reduced its membership to seven taxa (Guayasamin et al. 2009). Five species originally assigned to Cochranella were retained as incertae sedis within Centroleninae (i.e., “Cochranella’) due to a lack of molecular data and ambiguous behavioral and morpho- logical characters (“C.” balionota, “C.” duidaeana, “C.”
Correspondence. ‘ross@biodiversitygroup.org
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megista, “C.” riveroi, ‘C.” xanthocheridia, Guayasamin et al. 2009). Two of the latter species have since been shown to belong to the genus Nymphargus (Guayasamin et al. 2019; Trageser et al. 2021). Currently, eight species are recognized within Cochranella (Frost 2024), as well as two putative new species (Guayasamin et al. 2020). Among the lesser known members is the threatened Littoral Glassfrog, C. litoralis (Ruiz-Carranza and Lynch 1996). For this species, relatively few observations have been reported, the call and tadpole have yet to be described, and the evolutionary relationships among its congeners are still
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uncertain (Twomey et al. 2014; Guayasamin et al. 2020).
The reported distribution of C. /itoralis is restricted to lowland Chocoan rainforest below 250 m elevation in extreme southwestern Colombia and northwestern Ecuador (Ruiz-Carranza and Lynch 1996; Ruiz- Carranza et al. 1996; Grant and Morales 2010; IUCN SSC Amphibian Specialist Group 2019; Guayasamin et al. 2020). However, details in the literature regarding the extent of its distribution are ambiguous. While legitimate records have been reported from Narifio Department, Colombia, and Esmeraldas Province, Ecuador (Ruiz-Caranza and Lynch 1996; Guayasamin et al. 2006; Guayasamin et al. 2020; Pinto-Erazo et al. 2020), its distribution is also suggested to include Cauca Department, Colombia, and the Ecuadorian provinces of Los Rios, Pichincha, and Santo Domingo de los Tsachilas (Acosta-Galvis 2000; Lynch and Suaréz-Mayorga 2004; IUCN SSC Amphibian Specialist Group 2019; Guayasamin et al. 2020). The latter references appear to have either conflicting data therein or lack corroborating material, or both. As a result, the extent of its known distribution is unclear.
Here, the literature and available material for C. litoralis is reviewed to clarify its known distribution and produce an updated distribution map that reflects verifiable localities. In addition, two new localities are reported that extend its known distribution 175 km south- southeast and mark the highest documented elevation, its call is described for the first time, and its extinction risk is reassessed. Lastly, the call of C. granulosa is described from a recently documented population in Ecuador (Culebras et al. 2020), as available call analyses of this taxon are based on populations from Costa Rica and Panama (Ibafiez et al. 1999; Kubicki 2007).
Materials and Methods
Field work was conducted at two separate sites. The first stte was Los Laureles, Cotopaxi Province, Ecuador, where sampling efforts were conducted in March 2017 and March 2019. This area is characterized by a mosaic of cleared plots of land for agriculture and human settlements, with relatively small pockets of secondary forest. The second site was a fragmented forest near Cristobal Colon Quininde, Esmeraldas Province, Ecuador, where sampling was conducted in August 2021. The habitat consists of ca. 1.4 km? of secondary forest, with the northern end adjoining the Rio Canandé. Large forest clearings are present to the east, south, and west. Patches of cleared forest are also present north of the Rio Canandé, although intact mature forest is more prominent in this area and the protected forest of Reserva Biolégica Rio Canandé lies only about 3 km to the northwest, and Estacion Bioldgica Jevon is just to the northeast of the forest fragment. The plot of forest sampled was recently purchased to serve as a future rescue center and sanctuary for the Critically Endangered Ecuadorian Brown-headed
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Spider Monkey (Afeles f. fusciceps), and sampling of the biodiversity was conducted to generate a preliminary list of the taxa present in the forest.
Sampling was conducted using visual encounter surveys along trails and streams located within mature forest, disturbed forest, forest edge, and adjacent cleared areas, as well as agricultural plots. A Garmin 64s GPS receiver using WGS84 datum was used to collect geographic coordinates. Animals were verified as Cochranella litoralis using the diagnostic characters described in Ruiz-Carranza and Lynch (1996) and Guayasamin et al. (2020). Diagnostic photographs were taken of live specimens and submitted as vouchers to the digital repository at Centro Jambatu de Investigacion y Conservacion de Anfibios, San Rafael, Ecuador (CJ). Animals were returned to the exact location of capture after image and data collection, and released either the same night of capture or immediately at sunset the following evening to minimize stress. Field work was conducted under permit numbers 0013-18 IC-FAU- DNB/MA and MAE-ARSFC-2019-0163, authorized by the Ministerio del Ambiente del Ecuador, and carried out in accordance with the guidelines for the use of live amphibians and reptiles in field and lab research (Beaupre et al. 2004) compiled by the American Society of Ichthyologists and Herpetologists, the Herpetologists’ League, and the Society for the Study of Amphibians and Reptiles.
To assess and validate past records, we performed a search of the literature pertaining to C. /itoralis as well as various databases containing unpublished specimens or locality information. The literature search was conducted by entering key words from its taxonomic history into Google Scholar (1.e., “Centrolene litoralis,” “Centrolene litorale,” and “Cochranella litoralis’). Public-sourced and museum databases that were assessed include: Museo de Zoologia, Pontificia Universidad Catolica del Ecuador, Quito, Ecuador (QCAZ; https://bioweb. bio/faunaweb/amphibiaweb/), Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogota, Colombia (ICN; http://www.biovirtual.unal.edu.co/en/ collections/search/amphibians/),; VertNet (https://portal. vertnet.org); CalPhotos (https://calphotos. berkeley. edu); Naturalist (https:/(www.inaturalist.org); and HerpMapper (https://www.herpmapper.org). Confirmed localities were considered those that included a referenced specimen(s) or a combination of geographic coordinates and corroborating media. Extinction risk was assessed using the IUCN (2012) guidelines. Estimates for extent of occurrence (EOO) and area of occupancy (AOO) were calculated using the software GeoCAT (Bachman et al. 2011), following IUCN guidelines (IUCN 2022).
Bioacoustics. Call recordings for C. Jitoralis and C. granulosa were accessioned in the digital repository at Centro Jambatu de Investigacion y Conservacion de Anfibios, San Rafael, Ecuador (CJ). Call analyses for C.
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Ee Colombia
Fig. 1. Distribution map of Cochranella litoralis. White circles indicate verifiable localities reported in the literature; the white star denotes the type locality; the blue X marks the iNaturalist observation from the Rio Palenque Research Center, Los Rios; and yellow plus symbols indicate new records reported herein.
litoralis are based on four recordings of a single male (voucher CJ12588; call records: ec.cj.aud.26, ec.c). aud.28—30) obtained by JC on 16 March 2019 between 0400-0430 h after light rain. The recordings were taken at Los Laureles, Cotopaxi, Ecuador within an abandoned banana plantation next to a small patch of secondary forest. That area frequently floods after rains and has a small, shallow creek with slow moving water. One recording was made with an iPhone 7 in MPEG-4 format with a sampling rate of 44.1 kHz and 24-bits resolution. The other three were made with a Tascam DR-05 recorder in WAV format with a sampling rate of 44.1 kHz and 16- bits resolution. The iPhone 7 was placed approximately 3.5 m from the calling male and the Tascam DR-05 recorder was placed less than 0.5 m away.
The call analysis of C. granulosa is based on seven recordings (ec.cj.aud.27, ec.cj.aud.3 1-36) taken from two males obtained by JC on 16 March 2019 between 0315— 0340 h after light rain, and on 17 March 2019 between 2100-2115 h after light rain. The recordings were taken at Los Laureles, Cotopaxi, Ecuador, as reported by Culebras et al. (2020). The recordings were made with a Tascam DR-05 recorder in WAV format with a sampling rate of 44.1 kHz and 16-bits resolution. Recordings were made approximately 5 m from the calling male.
The Avisoft-SASLab Pro “Spectrogram tool” was used to analyze and filter the audio recordings. High resolution waveform, spectrogram, and power spectrum figures were generated using the R package Seewave (v2.2.1; Suer et al. 2008). To remove the background noise and facilitate the measurement of temporal and spectral parameters, a “Band Filter” was applied between 3,000—6,000 Hz and a “Noise Reduction” of 60 dB, with a threshold of -60 dB. The measurements were generated using Kaleidoscope Pro 5 software with the “Analyze View” tool, with a spectrogram configuration window
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of a 512-sample window size and 512 FFT size. The parameters assessed, as defined by Kohler et al. (2017), were dominant frequency (frequency with the most energy), bandwidth (difference between the upper and lower frequencies) and call duration (length of a note).
Results Cochranella litoralis (Ruiz-Carranza and Lynch, 1996)
New record. Adult male from Los Laureles, Cotopaxi, Ecuador (0°51718.2232” S, 79°11°25.926” W, 407 m; Fig. 1), 16 March 2019 at 0400 h; Christophe Pellet and Jaime Culebras leg.; photo voucher CJ12588 (Fig. 2); uncollected. The specimen was observed calling shortly after a light rain, perched on a leaf 4.5 m high within an abandoned banana plantation adjacent to secondary forest. Other males have been observed at this same location, the first being on 19 February 2017 at 2100 h. Males of Hyalinobatrachium tatayoi have also been observed calling nearby.
New record. Adult male, 20.6 mm snout-urostyle length (SUL), recorded from a fragmented forest adjacent to Cristobal Colon Quininde, Esmeraldas, Ecuador (0.45213°N, 79.14919°W, 178 m; Fig. 1); 21 August 2021 at 2310 h; Ross Maynard and Sebastian Kohn leg.; photo vouchers CJ12587a—d (Fig. 2); uncollected. The male was observed in a clearing 3 m from the forest edge, calling on the upper surface of a leaf within sparse herbaceous vegetation, perched 1.0 m high. Slow, shallow water was channeled just below the vegetation due to steady rain earlier that evening, which was flowing towards a small stream (about 2—3 m wide and 0.5 m deep) a few meters away. Two additional males were heard calling nearby, one from just within the forest and the other also in the clearing near the forest edge, however their exact locations were not observed. Other glassfrogs recorded along the stream adjacent to where the C. /itoralis was observed, but from within the secondary forest, were Sachatamia ilex and Teratohyla spinosa.
Distribution. A review of the literature yielded seven verified localities for C. Jitoralis: two localities from Colombia in extreme southwest Narifio Department, and five localities in Esmeraldas Province, Ecuador (Table 1; Fig. 1). The purported localities in Cauca, Colombia, and in Los Rios, Pichincha, and Santo Domingo de los Tsachilas, Ecuador, are either unverified or were reported in error (see Discussion). Nonetheless, a search of public-sourced and museum databases identified an additional locality from Los Rios Province, Ecuador, at the Rio Palenque Research Center in August 2021 (http://1Naturalist.org/ observations/90596035; D. Weaver and E. Osterman, pers. comm.). Since the coordinates of the holotype provided by Ruiz-Carranza and Lynch (1996) are imprecise, the placement of the type locality on the map 1s approximated (Fig. 1). The new record from Los Laureles, Cotopaxi,
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expands the elevational range from near sea level to 407 m asl, and extends the known distribution of C. /itoralis by abount 175 km south-southeast from the previous southernmost locality at Tsejpu, Rio Zapallo, Esmeraldas.
Extinction risk. Despite the new records, the extinction risk for C. /itoralis remains relatively high. With the additional localities reported herein, and assuming each of the seven localities where the species had previously been reported represent extant populations, the extent of occurrence (EOO) of the species is about 8,308 km? and the area of occupancy (AOO) is 40 km*. However, the only other reported observations over the past decade are from Tundaloma Lodge, Esmeraldas, Ecuador in 2014 (Guayasamin et al. 2020) and Tumaco, Narifio, Colombia, in 2015, 2016, and 2020 (Table 1; IUCN SSC Amphibian Specialist Group 2019; Pinto-Erazo et al. 2020; iNaturalist. org). Except for the latter locality in Colombia, whether there have been subsequent sampling efforts for C. /itoralis at the remaining localities in Esmeraldas Province, Ecuador is unclear. Although the status of these subpopulations cannot be verified at this time, we suspect that there has been recent and ongoing decline in the extent and quality of its habitat, given that northwest Ecuador has been a hotspot of deforestation over the past three decades (Sierra 2013; Kleeman et al. 2022). Logging and agriculture are
the main drivers of deforestation in the region, which have resulted in severely fragmented forests throughout its range. As a result of these ongoing pressures, C. /itoralis is currently known only from threat-defined locations (sensu IUCN 2012, 2022). While the observations reported here are the first to suggest that the species can tolerate altered habitat adjacent to forest, at least to some degree, the natural history and habitat requirements of the species remain poorly understood. Accordingly, and like the recent threat assessment for its national status in Ecuador (Ortega- Andrade et al. 2021), we recommend a global threat status of Endangered (EN) for C. /itoralis following IUCN criteria B2ab(111).
Call analysis. The call of C. /itoralis consists of a short, single tonal note (Fig. 3). The call duration was 88.51— 177.17 ms (& = 132.84 + 44.33; N = 4), the dominant frequency ranged from 5,210—5,304 Hz (x = 5,257 +47; N = 4), and the call bandwidth ranged from 738—1,729 Hz (x = 1,265 + 527; N = 4).
Compared to the available call descriptions of other species in the genus, C. nola and C. mache have similar call structures and parameter metrics. While C. nola exhibits a simple, non-pulsed note with comparable metrics (call duration: X = 95 ms + 11.97, dominant frequency: Xx = 5,460 Hz + 221; Létters and Kohler 2000; Kohler et al.
Fig. 2. Dorsal and ventral aspects of Cochranella litoralis in life. (A) Adult male, CJ12588, from Los Laureles, Cotopaxi, Ecuador. (B-E) Adult male, CJ12587a—d, from Cristobal Colon Quininde, Esmeraldas, Ecuador. Photos by: Jaime Culebras (A); Ross J.
Maynard (B-E).
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Fig. 3. Audio spectrogram (top), oscillogram (middle), and power spectrum (bottom) of an adult male Cochranella litoralis from Los Laureles, Cotopaxi, Ecuador (A), and an adult male C. granulosa from Jardin de los Suefios, Cotopaxi, Ecuador (B).
2006), C. mache has a call with two pulsed notes, a call duration of xX = 38 ms + 8, and a dominant frequency of xX = 5,410.2 Hz + 17.9 (Ortega-Andrade et al. 2013). Other Cochranella spp. that have described calls, such as C. granulosa (Fig. 3), have pulsed notes.
The call metrics measured from seven call recordings of C. granulosa observed at Jardin de los Suefios, Cotopaxi, Ecuador are as follows. Calls consisted of 1-4 notes ( X = 2.34), with 8-15 pulses per note; the first notes in multi-note calls are more pulsated than subsequent notes (first note: x = 15 + 2 pulses; second note: X = 12 + 2 pulses; third note X = 8 + 5 pulses); call duration was 150—1,437 ms ( X = 790 ms + 640); single-note duration varied from 130—260 ms, with the first note generally being longer than subsequent notes, similar to the calls of individuals from Costa Rica (Ibafiez et al. 1999; Kubicki 2007). The note interval was 45.49-179.31 ms (X = 112 + 66.91), and the dominant frequency was measured at 3,943-4,119 Hz (x = 4,031 + 88). Comparable metrics can be found in C. guayasamini, as it also exhibits a high-pitched, pulsed trill with two notes, with the first note having substantially more pulses than the second note (Twomey
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et al. 2014). Similar to the lack of phenotypic variation observed between populations of C. granulosa in Ecuador and Central America (Culebras et al. 2020), call variation also appears to be minimal.
Discussion
Although information for C. /itoralis remains limited, this study contributes new locality records and the first call analysis of this species, as well as that of C. granulosa from Ecuador. Our observation from Los Laureles, Cotopaxi, and the observation identified in the iNaturalist database from the Rio Palenque Research Center, Los Rios, are the first verified records outside of either Narifio Department, Colombia, or Esmeraldas Province, Ecuador. Other works that suggest its distribution includes Cauca Department, Colombia, and the Ecuadorian provinces of Pichincha, Santo Domingo de los Tsachilas, and Los Rios seem to do so in error or cannot be confirmed. Acosta-Galvis (2000) was the first to report the species from Cauca, but this was a mistake when citing the original description by Ruiz- Carranza and Lynch (1996). Lynch and Suaréz-Mayorga (2004) inexplicably report Guapi, Cauca as the species’
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only locality in Colombia, while omitting the type locality of La Guayacana, Narifio. Considering that there appears to be no evidence to support that locality, we suspect the former error led to the latter. The most recent Red List assessment for C. /itoralis also includes the Guapi locality, but the uncertainty of that locality is acknowledged (IUCN SSC Amphibian Specialist Group 2019). Notably, the only observations of C. /itoralis from Colombia since it was described were reported from adjacent to the type locality in Narifio (Pinto-Erazo et al. 2020), whereas no localities have been reported from Cauca.
The available information for populations within Ecuador is also confusing. The Red List assessment states that C. litoralis is known from the provinces of Esmeraldas, Santo Domingo de los Tsachilas, and Los Rios, however, the range map and extent of occurrence (EOO) exclude the latter two provinces (IUCN SSC Amphibian Specialist Group 2019). Although it is unclear why these provinces are mentioned, Cisneros-Heredia and McDiarmid (2007) suggested that two specimens collected in 1979 from the Rio Palenque Research Center, Los Rios, represent an undescribed taxon that is morphologically similar to C. litoralis. That locality lies on the border of Los Rios and Santo Domingo de los Tsachilas, which may have led to the confusion. Cisneros-Heredia and McDiarmid (2007) posit that the Rio Palenque specimens are distinguishable from C. litoralis based on a difference in iris coloration—unique red marks and reticulations as opposed to a salmon iris—but such variation is evident in images of C. /itoralis throughout much of its known distribution, including from the vicinity of the Rio Palenque Research Center (Fig. 2; 1Naturalist. org; Cisneros-Heredia and McDiarmid 2007; Guayasamin et al. 2020). Therefore, we believe it is unlikely that the specimens from the Rio Palenque Research Center are distinguishable from C. /itoralis, and, if true, that the taxon has been observed at this site in January 1979 and August 2021 (https://www.inaturalist.org/observations/90596035; https://collections.nmnh.si.edu/search/herps/; — Cisneros- Heredia and McDiarmid 2007).
Guayasamin et al. (2020) provide five localities with referenced vouchers and geographic coordinates for C. litoralis from Ecuador (see subsections “Specimens examined” and “Localities from the literature” therein). These data conflict with the localities depicted in the associated distribution map in both geographic position and number of localities, so we view these localities from the map as either unconfirmed or reported in error (Guayasamin et al. 2020). Guayasamin et al. (2020) also informally mentioned the presence of C. Jitoralis at Jardin de los Suefios, Cotopaxi, presumably based on an observation uploaded to the iNaturalist database by one of the authors of this paper (CP). However, and as we report herein, the observations from this area are not from the latter site, but instead from the nearby site of Los Laureles, Cotopaxi.
Considering that the conservation status of C. litoralis is primarily based on distribution data (IUCN
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SSC Amphibian Specialist Group 2019), our review of past localities along with the new records reported here provides an updated basis from which its extinction risk can currently be assessed. Like prior reports of this species, our observations are not from within protected areas. Nonetheless, the observation from Los Laureles, Cotopax1, was made about 2 km from the private reserve of Jardin de los Suefios, and is the second substantial range extension of a glassfrog discovered from the area (Culebras et al. 2020). Although the observation from Cristobal Colon Quininde, Esmeraldas, is only 3 km south of Reserva Bioldgica Rio Canandeé, there have surprisingly been no observations of C. litoralis documented there, although this area has been fairly well sampled (Mite et al. 2013).
Overall, the call analyses we provide improve our understanding of the natural history of these taxa, and can benefit efforts in field detection and studies of their respective species boundaries. Nonetheless, these glassfrogs remain poorly understood and there is little data to inform population trends either locally or across their known distributions. Future efforts are needed to fill these knowledge gaps, especially in light of the ongoing, broad- scale declines in the forest ecosystems from which they are known (Sierra 2013; Kleeman et al. 2022).
Acknowledgments. The authors thank Luis Coloma and Andrea Teran- Valdez at Centro Jambatu de Investigacion y Conservacion de Anfibios for permit acquisition and their assistance with accessioning our observations in the digital repository, and Andrés Mauricio Forero-Cano and David Weaver for information on their respective observations in the iNaturalist database.
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(Taylor, 1949) (Anura, Centrolenidae) found in Ecuador. Herpetology Notes 13: 353-355.
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Guayasamin JM, Cisneros-Heredia DF, Yanez-Mufioz MH, Bustamante MR. 2006. Notes on geographic distribution. Amphibia, Centrolenidae, Centrolene ilex, Centrolene litorale, Centrolene medemi, Cochranella albomaculata, Cochranella ametarsia. Range extensions and new country records. Check List 2: 24-25,
Guayasamin JM, Castroviejo-Fisher S, Trueb L, Ayarzaguena J, Rada M, Vila C. 2009. Phylogenetic systematics of glassfrogs (Amphibia: Centrolenidae) and their sister taxon A/lophryne ruthveni. Zootaxa 2100: 1-97.
Guayasamin JM, Cisneros-Heredia DF, McDiarmid RW, Pefia P, Hutter CR. 2020. Glassfrogs of Ecuador: diversity, evolution, and conservation. Diversity 12(6): 222)
Ibafiez DR, Rand AS, Jaramillo A. 1999. Los Anfibios del Monumento Natural Barro Colorado, Parque Nacional Soberania y Areas Adyacentes. Editorial Mizrachi y Pujol, Santa Fe de Bogota, Colombia. 187 p.
IUCN SSC Amphibian Specialist Group. 2019. Cochranella litoralis. The IUCN Red List of Threatened Species 2019: e.T54923A49367704.
IUCN Standards and Petitions Committee. 2022. Guidelines for Using the IUCN Red List Categories and Criteria. Version 15.1. Prepared by the Standards and Petitions Committee, International Union for the Conservation of Nature, Gland, Switzerland. 114 p.
Kleeman J, Zamora C, Villacis-Chiluisa AB, Cueca P, Koo H, Noh JK, Furst C, Thiel M. 2022. Deforestation in continental Ecuador with a focus on protected areas. Land 11(268): 1-26.
Kohler J, John A, Bohme W. 2006. Notes on amphibians recently collected in the Yungas de La Paz region, Bolivia. Salamandra 42(1): 21-27.
Kohler J, Jansen M, Rodriguez A, Kok PJ, Toledo LF, Emmrich, M, Vences M. 2017. The use of bioacoustics in anuran taxonomy: theory, terminology, methods, and recommendations for best practice. Zootaxa 4251(1): 1-124.
Kubicki B. 2007. Ranas de Vidrio de Costa Rica/Glass Frogs of Costa Rica. Editorial INBio, Santo Domingo de Heredia, Costa Rica. 304 p.
Lotters S, Kohler J. 2000. Cochranella nola (Anura: Centrolenidae): natural history notes, distribution, and advertisement call. Herpetological Natural History 7(1): 79-81.
Lynch JD, Suarez-Mayorga A. 2004. Catalogo de anfibios
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en el Choco Biogeografico. Pp. 654—668 In: Colombia Diversidad Biotica IV, El Choco Biogeogrdafico/ Costa Pacifica. Volumen I. Editor, Orlando Rangel C. Universidad Nacional de Colombia, Bogota, Colombia. 862 p.
Mite MAM, Yanez-Mufioz MH, Meza-Ramos PA, Reyes- Puig MA. 2013. Reserva Biologica Rio Canandé. Entre los Ultimos relictos de bosque hiumedo Tropical en la Costa. In: Herpetofauna en Areas Prioritarias para la Conservacion: el Sistema de Reservas Jocotoco y Ecominga. Serie de Publicaciones del Museo Ecuatoriano de Ciencias Naturales, Monografia 6. MECN, Fundacion para la Conservacion Jocotoco, Fundacion Ecominga, Quito, Ecuador. 392 p.
Ortega-Andrade HM, Rojas-Soto O, Paucar C. 2013. Novel data on the ecology of Cochranella mache (Anura: Centrolenidae) and the importance of protected areas for this Critically Endangered glassfrog in the Neotropics. PLoS ONE 8(12): e0081837.
Ortega-Andrade HM, Blanco MR, Cisneros-Heredia DF, Arévalo NG, Vargas-Machuca KGL, Sanchez- Nivicela JC, Armijos-Ojeda D, Andrade JFC, Reyes- Puig C, Riera ABQ, et al. 2021. Red List assessment of amphibian species of Ecuador: a multidimensional approach for their conservation. PLoS ONE 16(5): e0251027.
Pinto-Erazo MA, Calderén-Espinosa ML, Medina- Rangel GF, Galeano MAM. 2020. Herpetofauna de dos municipios del suroeste de Colombia. Biota Colombiana 21(1): 41-57.
Ruiz-Carranza PM, Lynch JD. 1996. Ranas Centrolenidae de Colombia IX. Dos nuevas especies del suroeste de Colombia. Lozania Acta Zooldgica Colombiana 30(68): I-11.
Ruiz-Carranza PM, Ardila-Robayo MC, Lynch JD. 1996. Lista actualizada de la fauna de Amphibia de Colombia. Revista de la Academia Colombiana de Ciencias Exactas, Fisicas, y Naturales 20(77): 365-415.
Sierra R. 2013. Patrones y Factores de Deforestacion en el Ecuador Continental, 1990-2010, y un Acercamiento a los Proximos 10 Afios. Conservacion Internacional Ecuador / Forest Trends, Quito, Ecuador. 45 p.
Suer J, Augin T, Simonis C. 2008. Seewave, a free modular tool for sound analysis and synthesis. Bioacoustics 18(2): 213-226.
Taylor EH. 1951. Two new genera and a new family of tropical American frogs. Proceedings of the Biological Society of Washington 64: 33-40.
Trageser SJ, Maynard RJ, Culebras J, Kohn S, Quezada A, Guayasamin JM. 2021. Phylogenetic position of the glassfrog “Cochranella” megista (Anura: Centrolenidae) and first records for Ecuador. Phyllomedusa 20: 27-35.
Twomey EM, Delia J, Castroviejo-Fisher S. 2014. A review of northern Peruvian glassfrogs (Centrolenidae), with the description of four new remarkable species. Zootaxa 3851: 1-87.
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Ross Maynard is a biologist and photographer for The Biodiversity Group in Tucson, Arizona, USA, and serves as Director of Research for their biodiversity and conservation projects in Ecuador. His education includes a B.S. in Zoology (North Carolina State University) and an M.Sc. in Biology (Stephen F. Austin State University, Nacogdoches, Texas, USA), and his research interests center on the conservation, ecology, and diversity of amphibian and reptile assemblages. His efforts have primarily been focused in Ecuador since 2007, but he has also worked in regions of Mexico, Vietnam, and Bolivia. Ross also serves as a Contributor for the IUCN SSC Amphibian Specialist Group, and has served on the Board of Directors of the Tucson Herpetological Society since 2017.
José Tinajero-Romero is a biologist interested in bioacoustics, conservation, and ecology. His Bachelor’s thesis compared the different ensembles in a community of bats in Bosque Cerro Blanco dry forest in Ecuador, using echolocation as a method for identification and collecting data. He has participated mostly 1n projects that involve bat echolocation and ecology, but also has experience with bioacoustics in other taxonomic groups such as amphibians and birds.
Sebastian Kohn holds a Bachelor of Arts (B.A.) degree in Biology and Environmental Studies from Whitman College, Walla Walla, Washington, USA. Sebastian has experience in community wildlife management and postgraduate courses in Planning and Development of Sustainable Development projects, with a focus on biodiversity and sustainable agriculture, at Stellenbosch University in South Africa. He has been leading conservation and research efforts in Rio Manduriacu Reserve in Ecuador since 2008, as well as researching Andean Condors since 2012 and the Black-and-Chestnut Eagle since 2018. Sebastian is currently the Executive Director of Fundacion Condor Andino, an Ecuadorian NGO focused on the research and conservation of endangered species in this megadiverse country.
Christophe Pellet is an ardent environmentalist who founded the conservation project “Bosque protegido El Jardin de los Suefios” in Ecuador with the aim of preserving biodiversity and raising awareness of the importance of remote forests and the ecosystem services they provide. He dedicates his time and energy to the conservation of biodiversity in the canton of La Mana, Ecuador, while sharing his knowledge with local communities through environmental education programs.
Jaime Culebras was born in Caceres, Spain, and has an M.Sc. in Environmental Education and an M.Sc. in Biodiversity and Conservation of Tropical Areas. Jaime has been living in Ecuador for more than 13 years, where he works as a reptile and amphibian researcher and nature photographer. Jaime has co-authored several papers on the biogeography, natural history, and descriptions of new species. He has published in international magazines such as National Geographic, and received numerous international photography and conservation awards such as World Press Photo, Wildlife Photographer of the Year, and Montphoto. He is a co-founder of Photo Wildlife Tours and a research associate of Fundacion Condor Andino. His greatest interests are publicizing the existence and importance of threatened species, promoting love towards reptiles and amphibians, and fighting against nature crimes.
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Official journal website: amphibian-reptile-conservation.org
Amphibian & Reptile Conservation 18(1&2) [General Section]: 10-19 (e330).
Evaluation of p-Chip microtransponder tags on small-bodied salamanders (Eurycea spp.)
1"Desiree M. Moore, ?7Madeleine S. Gillis, and *Thomas S. Funk
'United States Fish and Wildlife Service, San Marcos Aquatic Resources Center, 500 East McCarty Lane, San Marcos, Texas 78666, USA *Student Conservation Association, San Marcos Aquatic Resources Center, 500 East McCarty Lane, San Marcos, Texas 78666, USA *United States Fish and Wildlife Service, Inks Dam National Fish Hatchery, 345 Clay Young Road, Burnet, Texas 78611, USA
Abstract.—Reliable approaches for tracking individual organisms are needed for research purposes and to inform the conservation and management of aquatic organisms. However, safe and dependable tagging methods are difficult to implement for small-bodied organisms. The objective of this study was to examine survival, tag retention, and growth in three aquatic salamander species of different sizes (Barton Springs Salamander, Eurycea sosorum; Comal Springs Salamander, Eurycea pterophila; Texas Blind Salamander, Eurycea rathbun)i) injected with p-Chip tags in a captive setting. The ability of novice scanners to read p-Chips over the duration of the study was also assessed. Post-tagging survival was high across all treatments for all species (97-100%). Tag retention among species was similar (97—100%), and growth appeared unaffected by tagging. No relationship between success of tag readability and time since tagging was found, and all novice scanners were able to read the tags implanted in 100% of Comal Springs and Texas Blind Salamanders. However, variability was found with novice scanners reading tags in Barton Springs Salamanders, although all tags were successfully read by an experienced scanner. P-Chips provided an improved readability rate, reduced human error, and allowed for more individual identification codes than the visible implant elastomer tags commonly used for these species. This study shows that p-Chips are suitable tags for small-bodied aquatic salamanders.
Keywords. Eurycea sosorum, Eurycea pterophila, Eurycea rathbuni, retention, survival, tracking
Citation: Moore DM, Gillis MS, Funk TS. 2024. Evaluation of p-Chip microtransponder tags on small-bodied salamanders (Eurycea spp.). Amphibian & Reptile Conservation 18(1 & 2) [General Section]: 10-19 (e330).
Copyright: © Moore et al. 2024. This is an open access article distributed under the terms of the Creative Commons Attribution License [Attribution 4.0 International (CC BY 4.0): https://creativecommons.org/licenses/by/4.0/], which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The official and authorized publication credit sources, which will be duly enforced, are
as follows: official journal title Amphibian & Reptile Conservation; official journal website: amphibian-reptile-conservation.org.
Accepted: 7 February 2024; Published: 11 August 2024
Introduction
Wildlife tagging provides a reliable method for the identification and differentiation of individuals that would otherwise be challenging to distinguish, and it allows for tracking a variety of life history parameters (Ricker 1956). In the field, mark-recapture efforts can provide information about growth, survival, habitat range, migration, age, condition, and other key parameters (Ricker 1956; Silvy et al. 2005; Osbourn et al. 2011; Moon et al. 2022) and marked organisms can be monitored over time in individuals, cohorts, and subsets of populations and communities. In captive settings, tagging eliminates the need to separate organisms or cohorts into different tanks or enclosures for identification, allowing enclosures to be stocked to capacity, thereby conserving space. It also facilitates the tracking of rare occurrences in captive individuals, such as reproductive events or illness. However, tags
Correspondence. ‘desiree_moore@fws.gov
Amphib. Reptile Conserv.
that are not compatible with a species can cause injury, mortality, or behavioral changes in the organism due to stress and difficulty in functioning normally (Musselman et al. 2017; Moon et al. 2022). In these cases, the results of any tagging program would be negatively affected by the tagging method.
A long and growing list of wildlife marking methods exists, allowing researchers to tailor methods to their study needs while accounting for the organism’s biology and life history (Silvy et al. 2005). Tags should be selected to maximize various factors such as tag retention and longevity, cost efficiency, and ease of use while minimizing stress, handling time, and other factors that negatively affect growth, behavior, and survival (Osbourn et al. 2011). Failure to mitigate these negative effects can violate mark-recapture assumptions (Ricker 1956) and tagging ethics (Cooke et al. 2013), preventing the extrapolation of meaningful conclusions from data (Murray and Fuller 2000). General amphibian
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tagging practices include digit clipping (Phillott et al. 2007; Waddle et al. 2008), branding (Donnelly et al. 1994; Measey et al. 2001), affixing or implanting radio transmitters (Weick et al. 2005), staining (Carlson and Langkilde 2013; Fischer et al. 2020), pattern mapping and software assisted photo identification (Andreone 1986; Gamble et al. 2008; Bendik et al. 2021), injecting elastomers, and attaching transponders (Sinsch 1997; Donnelly et al. 1994; Moon et al. 2022).
Central Texas is populated by several species of endemic, paedomorphic salamanders of state and federal conservation concern with narrow distributions and poorly understood demographies. Among these are the state and federally endangered Texas Blind Salamander (Eurycea rathbuni) and Barton Springs Salamander (Eurycea sosorum), and a population of the non-listed Fern Bank Salamander from Comal Springs (reclassified from Eurycea neotenes to Eurycea pterophila by Devitt et al. 2019). The Barton Springs Salamander is protected under the Barton Springs Habitat Conservation Plan (Barton Springs/Edwards Aquifer Conservation District 2018), and the Texas Blind Salamander and Comal Springs Salamander are protected under the Edwards Aquifer Habitat Conservation Plan (Edwards Aquifer Authority 2012). Tagging these animals requires accommodating their small size and permeable skin (Heemeyer et al. 2007). This is achieved by selecting small tags with a high tag retention rate associated with that species. Additionally, it is important to minimize mortality when working with threatened and endangered species, so stress and handling time should be considered.
Few studies have used tagging techniques specifically on either Barton Springs, Texas Blind, or Comal Springs Salamanders. Bendik et al. (2021) successfully employed photographic identification software to track Barton Springs Salamanders without placing a tag. However, when using photo identification in Jollyville Plateau Salamanders (Eurycea tonkawae), another paedomorphic salamander species found in Central Texas, misidentification rates slowly increased over time in adults and rapidly increased (within 2 months) in juveniles due to growth-associated pigment changes (Bendik et al. 2013). Passive Integrated Transponder (PIT) tags, bio-compatible glass-encased microchips typically ranging in size from 8-14 mm (Gibbons et al. 2004), yielded a low retention rate in adult Texas Blind Salamanders (Moon et al. 2022). PIT tags are best suited for larger individuals and species over 50 mm in standard length (Musselman et al. 2017), rendering them unsuitable for juveniles and smaller species. Visible Implant Alphanumeric (VIA) tags, small (1.2 mm x 2.7 mm to 2.0 x 5.0 mm) fluorescent plastic tags printed with visible 3-digit alphanumeric codes (Northwest Marine Technology Inc. 2019), were rejected by Comal Springs Salamanders by tearing through the skin and falling out through the resulting wound (Moon et al.
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2022). Even when salamanders successfully retained VIA tags (e.g., Texas Blind Salamanders; Moon et al. 2022), reading was inhibited when the tag was injected too deeply beneath an insufficiently translucent tissue (Osbourn et al. 2011), or at an angle to the epidermal surface (Moon et al. 2022). Visible Implant Elastomer (VIE) tags, injectable colored liquid tags that cure into flexible, fluorescent, bio-compatible solids (Northwest Marine Technology Inc. 2019), exhibited great ease of use and retention in Texas Blind and Comal Springs Salamanders (Moon et al. 2022) and had no observable effects on survival or growth in San Marcos salamanders (Eurycea nana) (Phillips and Fries 2009). VIE tags are injected with a 29-gauge needle (Davis and Ovaska 2001), making them suitable for small salamanders. Tag reading success was generally high (Moon et al. 2022), but has been reported to be reduced in some cases by the propensity of the tags to break, degrade, and migrate (Heemeyer et al. 2007). Additionally, certain elastomer colors are difficult to discern even by a trained eye (DMM, TSF, pers. obs.; Northwest Marine Technology Inc. 2021), occasionally resulting in human error. As a result, the sparse color palette available limits the number of differentiable markings possible (< 10) unless using multiple color injections, which increases handling time (Davis and Ovaska 2001), requires additional wounds, and exacerbates stress.
As arelatively new technology, p-Chips are injectable microtransponders with a unique set of characteristics that present an alternative option for tagging small- bodied species, including neotenic salamanders. Tag detection and reading is accomplished with a laser wand that transmits information in the form of a 9-digit serial number from the p-Chip photocells to a computer using specialized software (PharmaSeq, Princeton, New Jersey, USA), which expedites tag detections and readings and eliminates the potential for human error. The small size of p-Chips (500 um x 500 um x 100 um) renders them nearly invisible and should not provoke social (Fiske 1997; Frommen et al. 2015; Fischer et al. 2020) or predatory (Catalano et al. 2001; Carlson and Langkilde 2013) behavioral responses from surrounding animals, which are sometimes associated with colored tags. P-Chips have shown high tag retention (Chen et al. 2013; Moore and Brewer 2021) and subject survival rates (Faggion et al. 2020; Moore and Brewer 2021) in studies performed on small-bodied fishes. Thus, p-Chips could be effective for other small-bodied aquatic species, including salamanders.
To our knowledge, p-Chip microtransponder tags have not yet been tested in salamanders or any other amphibians. The purpose of this study was to examine the efficacy of p-Chips in small-bodied aquatic salamanders by measuring survival and tag retention in three salamander species of different sizes (Eurycea sosorum, Eurycea pterophila, and Eurycea rathbuni) injected with p-Chip tags in a captive setting.
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Materials and Methods
All salamanders used in this study, namely Barton Springs Salamanders (Eurycea sosorum, n = 95), Comal Springs Salamanders (Eurycea pterophila, n= 111), and Texas Blind Salamanders (Eurycea rathbuni, n = 78), were part of the captive-assurance populations (1.e., Critically Endangered and threatened animals in captivity for reintroduction purposes) located at the United States Fish and Wildlife Service San Marcos Aquatic Resources Center in San Marcos, Texas, USA. When possible, we prioritized using captive-bred salamanders to minimize potential harm to the wild stock population. However, due to the limited availability of captive-bred individuals, 51 captive-held wild stock Texas Blind Salamanders were included. All salamanders in this study were adults except for 19 juvenile captive-bred Texas Blind Salamanders. Adult salamanders were held in seven tanks, each with a volume of approximately 265 L (70 gal), maintained at a depth of 23 cm. Each tank of adult salamanders contained one species of salamander and was divided into equal sections with water-permeable barriers to separate the treatment groups while maintaining controlled environmental conditions across the groups. Juvenile salamanders were held in three 38 L (10 gal) aquaria with an 18 cm water depth. Each aquarium contained one treatment group, but all aquaria received water from the same source to keep the water quality consistent. Tanks were supplied with flow-through well water at a temperature of 20-23 °C. Each tank section and aquarium had a similar assortment of habitat structures (rocks, aquarium plants, etc.). Adult salamanders were fed live blackworms and live Daphnia once weekly, and live Daphnia and frozen Mysis once weekly. Additionally, because of their larger size, adult Texas Blind Salamanders were fed live red worms (Eisenia fetida, Eisenia hortensis, and Perionyx excavatus) cut into small pieces each week. Juvenile Texas Blind Salamanders were fed live Artemia and Daphnia twice weekly. All feeds except for the red worms were supplied at a portion of 0.25 mL/salamander. Red worms were supplied at a portion of 1.6 cm/salamander. Tanks were cleaned weekly.
Prior to launching the full study, we ran a pilot study with five salamanders of each species tagged using the methods described below and monitored for one month to assess potential mortality in the federally listed species. All salamanders survived and retained their tags. Pilot study salamanders were not used in any analyses.
Salamanders were anesthetized before being placed into treatment groups, and measurements were taken. Salamanders were anesthetized via immersion in tricaine methanesulfonate (MS-222, 0.5 g/L) buffered with sodium bicarbonate using previously established protocols (Wright 2001). The salamanders were randomly assigned into treatment groups (tagged, sham, and control) using a random number generator. Sample size varied among treatments due to the limited availability of salamanders (Table 1). The tagged groups were the most numerous for
Amphib. Reptile Conserv.
Fig. 1. The left side of a gravid female salamander. The black arrow points to the p-Chip tag.
each species to ensure the validity of statistical analyses examining tag retention. Salamanders were placed in clear re-sealable, sliding channel, polyethylene storage bags for easier handling (Heemeyer et al. 2007). Each salamander was measured for weight (g) and snout-vent length (SVL, mm; Petranka 1998; Table 1), sexed using the candling method (Gillette and Peterson 2001), and any distinguishing features or behaviors were recorded (e.g., the presence of eggs or regurgitation). Technical difficulties prohibited the weighing of 40 of the 95 Barton Springs Salamanders (Table 1).
Three treatment groups were used to examine the effects of p-Chips on salamander survival. Following the manufacturer’s guidelines (Pharmaseq Inc. 2020), a 0.8 mm diameter hypodermic needle was used to inject a 500 um x 500 um x 100 um p-Chip subcutaneously at the base of the tail just dorsal and posterior to the left hindlimb of each salamander in the tagged groups (Fig. 1). After tagging, the p-Chips were scanned with the laser reader to record the unique 9-digit tag number. Sham salamanders were treated the same as tagged salamanders (e.g., handled and punctured with the needle) except no tag was placed. Control salamanders were handled the same as the tagged and sham salamanders but were not tagged or pierced with a needle. Using both sham and control groups allowed us to distinguish between the effects of the handling process and the effects of the tag itself (Jepsen et al. 2015; Moore and Brewer 2021). After handling, the salamanders were placed in a small recovery tank until they were able to right themselves and swim normally. Salamanders were then moved to the appropriate section of their holding tank. For consistency, one researcher (DM) performed all the tagging.
Salamander survival, tag retention, and tag readability were monitored for eight months, and growth was examined at the conclusion of the study. Survival was monitored daily as part of the normal husbandry care. Each week, a researcher with experience in scanning p-Chips (DM) scanned all tagged salamanders to check
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Tagging small-bodied salamanders (Eurycea spp.)
retention. Tags were considered lost if the scanner could not detect a tag for the remainder of the trial. Tag readability, 1.e., the ability to obtain the identification code of an implanted p-Chip tag, was assessed over time by novice scanners monthly. A novice scanner, 1.e., someone who never scanned or had experience with p-Chips before participating in this study, scanned a subset of at least 20% of the salamanders each month to assess any tag readability differences between the experienced and novice scanners. The subset was selected by randomly selecting a tank tagged group and requiring the novice scanner to scan all individuals in that group. A new novice scanner was used each month to examine readability across many individuals and avoid bias due to any experience gained over the duration of the study. Readability was quantified as the percentage of salamanders successfully scanned by the novice scanner. Salamanders were not anesthetized during scanning events to reduce unnecessary stress. There were not enough participants to have a novice scanner for each species each month; so, the order in which salamander species were scanned varied across novice scanners to reduce bias due to any experience gained during the scanning process. SVLs were recorded at the conclusion of the study to determine if growth was affected by tagging, although final weights were not recorded to reduce unnecessary salamander stress.
Analyses. Kaplan-Meier time-at-event curves (Goel et al. 2010) were built to examine survival and retention over time. These curves estimated the probability of an event (survival or retention) occurring at each time interval. Days post tagging and weeks post tagging were used as the time increments for survival and retention, respectively. This approach could reveal any differences across time that may be missed with other methods. For example, high mortality immediately following tagging might be an indication of harm from tagging even if survival rates are somewhat similar across groups. The two null hypotheses tested using log-rank tests were that survival curves did not differ among treatments or by sex for each species and that retention curves did not differ among species. Only salamanders that could be sexed were included in tests comparing survival between sexes (Table 1). Data for juvenile and adult Texas Blind Salamanders were pooled due to the similarity in results. Differences were considered significant at a < 0.05. Kaplan-Meier curves and log-rank tests were performed in the “survival” package (Therneau 2020) in the program R version 4.2.2 (R Core Team 2022).
Tag readability over time and the effects of tagging on growth were examined. Tag readability was assessed using Pearson’s pairwise correlation coefficient to determine the correlation between the percentage of scanned tags to the time since tagging in months. A correlation was considered to be strong at |r| > 0.50. One- way ANOVAs were performed to confirm that there were
Amphib. Reptile Conserv.
no differences in initial SVLs among treatments for each species. To determine the effects of tagging on growth, two-sample t-tests were conducted to compare the growth of tagged salamanders to control salamanders for each species. Growth was calculated by subtracting the initial SVL from the final SVL of each salamander, and differences were considered significant at a < 0.05. All assumptions for analyses (normality, homoscedasticity, independence, and no outliers) were met by the data in this study. The base package in the program R version 4.2.2 (R Core Team 2022) was used to calculate the correlation coefficients and conduct the t-tests.
Results
Tagging salamanders with p-Chips had no effect on their survival, and no difference in survival was evident between male and female salamanders (Figs. 2-4). Survival did not differ among treatment groups for Barton Springs Salamander (v7 = 0.5, p = 0.8), Comal Springs Salamander (y* = 2.3, p = 0.3), or Texas Blind Salamander (y? = 1.1, p = 0.6). Additionally, survival did not differ between males and females among the treatment groups for Barton Springs Salamander (y? 4.5, p = 0.5), Comal Springs Salamander (7 = 5.2, p = 0.4), or Texas Blind Salamander (y? = 2.4, p= 0.8). Three Barton Springs Salamander mortalities (two tagged and one control) occurred on day 138 of the study, and an additional mortality in the sham group occurred on day 177 (Fig. 2). One Comal Springs Salamander mortality occurred in the control group on day 150 (Fig. 3), and one adult Texas Blind Salamander mortality occurred in the tagged group on day 191 (Fig. 4). Tag retention was relatively high and did not differ among species (7 = 1.1, p= 0.06; Table 2). One Barton Springs Salamander tag was lost in week 1 of the study, and one tag in a Comal Springs Salamander was either lost or shifted to the point that it could not be read in week 6 of the study (Fig. 5).
Tag readability for Barton Springs Salamanders varied across novice scanners, but all Comal Springs Salamander and Texas Blind Salamander tags were readable by every novice (Table 3). There was nota strong correlation between readability and time since tagging for Barton Springs Salamanders over the eight months of this study (r = 0.31). Only the experienced scanner was able to read one of the Barton Springs Salamander tags. This salamander was randomly selected for reading by novice scanners in months 1-4 and 6. Novice scanners in months 3 and 4 were unable to read other salamander p-Chip tags, but those tags were successfully read by novice scanners thereafter (Table 3). The novice scanners in months 3 and 4 both attempted to read the Barton Springs Salamanders first and then read the other species’ tags afterward. All novice tag scanners were able to accurately read all Comal Springs Salamander and Texas Blind Salamander tags throughout the study.
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Control 0.80 0.90 1.00
1.00
Sham
0.80 0.90
Survival probability
Tagged 0.80 0.90 1.00
Fig. 2. Kaplan-Meier survival curves for Barton Springs Salamanders in the control, sham, and tagged groups. The probability of survival is shown with 95% confidence intervals (dashed lines) over time in days since tagging.
Control 0.80 0.90 1.00
0 50 100 150 200
> — ‘0 oe 8s Oo ™- o Eo age a? Oo a 3 T T T T T Pa 0 50 100 150 200 = “” oO oO 3 el Das DS Oo 00 Oo
0 50 100 150 200 Days
Fig. 4. Kaplan-Meier survival curves for Texas Blind Salamanders in the control, sham, and tagged groups. The probability of survival is shown with 95% confidence intervals (dashed lines) over time in days since tagging.
Growth was not affected by tagging in any of the three species. Initial lengths and weights were closely correlated (Pearson’s product moment coefficient |r| = 0.93), and so the lengths were used for growth analyses. The initial lengths did not differ among treatments for Barton Springs (F2, 92 = 1.39, p= 0.25), Comal Springs (F2, 108 = 0.39, p = 0.68), or Texas Blind (F2, 75 = 0.47, Pp = 0.63) salamanders. Although the final mean SVL was smaller than the initial mean SVL for some groups
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Control 0.80 0.90 1.00
> ~_ = oe & oO ™_ Se. om a —Y Goo 2 a T T T T T Pa 0 50 100 150 200 pm | n” oO 2 3 - QS oO oO f- (jo) ja T T T T T 0 50 100 150 200 Days
Fig. 3. Kaplan-Meier survival curves for Comal Springs Salamanders in the control, sham, and tagged groups. The probability of survival is shown with 95% confidence intervals (dashed lines) over time in days since tagging.
BSS 0.80 0.90 1.00
> = ‘OD § Oo 8 cS S a Wo Cc O66 ee ees ® 8g (o) , o>) a0) -
TBS 0.80 0.90 1.00
0 5 10 15 20 25 30 35 Weeks
Fig. 5. Kaplan-Meier p-Chip retention curves for Barton Springs Salamanders (BSS), Comal Springs Salamanders (CSS), and Texas Blind Salamanders (TBS). The probability of p-Chip retention is shown with 95% confidence intervals (dashed lines) over time in weeks since tagging.
(Table 1), growth did not differ between the tagged and control groups for Barton Springs Salamander (p = 0.84), Comal Springs Salamander (p = 0.53), or Texas Blind Salamander (p = 0.64). Visible growth was noted in the juvenile Texas Blind Salamanders, but not in any other groups. Although not formally examined, we noted that the control and tagged groups of Comal Springs and Texas Blind Salamanders produced multiple clutches of viable eggs over the duration of the study.
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Discussion
Improving tagging methods for small-bodied aquatic organisms is important for conservation and management, and progress is underway with new technological developments. P-Chips resulted in high survival (97-100%) and tag retention (97—100%) without inhibiting growth in aquatic salamanders, indicating the potential for using p-Chip tags in lab and field studies involving aquatic salamander species. Additionally, p-Chips provided an improved readability rate, reduced human error, and allowed for a greater number of individual identification codes than the VIE tags commonly used for these species. To our knowledge, this is the first study examining p-Chips in aquatic salamander species.
We found p-Chips to be appropriate and versatile tags for aquatic salamanders. P-Chips provided high survival and retention rates in Barton Springs, Comal Springs, and Texas Blind Salamanders. Our results are similar to those of studies examining p-Chips in other aquatic organisms (Chen et al. 2013; Faggion et al. 2020; Moore and Brewer 2021). Although photo identification has been used in Barton Springs Salamanders (Bendick et al. 2021), we are unaware of any other published studies examining the efficacy of photo identification in any of the species in our study. Photo identification is labor intensive as it requires time to take and process the photos. Tagging and scanning p-Chips requires only seconds for an experienced tagger. P-Chips would be preferred to photo identification when time is a concern and an experienced tagger is available. P-Chips were more versatile than the previously used VIE tags. Although survival and tag retention were similar in previous VIE tagging research (Phillips and Fries 2009; Moon et al. 2022), p-Chips provided individual identification with a single tag. To achieve the same resolution of individual information provided by a single p-Chip tag, especially in large sample sizes, VIE tag codes would require the injection of multiple tags per individual. Increasing the number of wounds might increase animal stress, the possibility of infection, and mortality over time. P-Chips also enabled individual identification in small (e.g., <35 mm SVL) salamanders that might not be able to survive the injection of several VIE tags. There were fewer opportunities for human error when using p-Chips because tag codes were recorded directly from the laser reader into a CSV file instead of being manually observed, interpreted, and written or typed. Additionally, novice scanners were more successful at reading p-Chips (100%) compared to novice scanners reading VIE tags in Comal Springs and Texas Blind salamanders (Moon et al. 2022). It is not currently known whether p-Chips would perform as well when applied to salamanders in a wild setting where habitats are more variable and predators might be present.
Amphib. Reptile Conserv.
Although tag readability was optimal for Comal Springs and Texas Blind Salamanders, novice scanners made occasional errors when reading p-Chips in Barton Springs Salamanders. Unlike with VIE tags in Eurycea spp. (Moon et al. 2022), tag readability in Barton Springs Salamanders was not related to time since tagging. Instead, the difficulty reading tags seemed to be related to individual salamander tag placement and variations in scanner ability, and was unique to the Barton Springs Salamanders in this study. For example, one individual salamander was unreadable by all novice scanners who attempted to scan it. The experienced scanner noted that the tag in this salamander was at an angle, and the tag had to be read by pointing the laser upward from the underside of the salamander. The novice scanners did not have the experience to identify and troubleshoot this issue and were unable to read that tag. This instance indicates that the experience of the tagger might be important for overall readability, since a less experienced tagger might not be able to tag as many individuals with consistent placement regarding depth, angle, and location. Another possibility 1s that readability may have been increased for the tagger, as hypothesized with VIE tags (Moon et al. 2022). The wide range of readability scores (50—100%) indicated that individual scanner variation might affect novice readability in Barton Springs Salamanders. Possible reasons for this variation include variation in eyesight, patience, interest, and similar experiences. For example, individuals that have read other types of tags in the past might be more able to read p-Chips without direct experience.
It is notable that readability issues were only present for Barton Springs Salamanders, indicating there might be some anatomical or behavioral traits that reduce readability overall. Another possibility is that this issue was partially due to novice scanners having more trouble with the first species they read. However, this issue was not seen for novice scanners that read other species first, and some scanners that began with other species were not able to read all the Barton Springs Salamander tags. On several occasions, the experienced and novice scanners noted that the Barton Springs Salamanders seemed more physically active than the other species, so it was difficult to keep them positioned long enough to find the proper angle for reading. Anesthetizing salamanders during the scanning event might improve the ability of novice scanners to read these tags. However, repeated anesthetization in these species is not well studied and might have negative effects. Additionally, the Barton Springs Salamanders are more pigmented than Texas Blind Salamanders and seem to have thicker skin than Comal Springs Salamanders, a trend that might be related to size (1.e., larger salamanders tended to have thicker skin during tagging). Future work should examine the differences among species and ontogenetic stages that could be contributing to this readability issue, which could indicate those species that are most suitable for using p-Chip tags.
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The data indicated no evidence that growth was affected by p-Chip tagging or contributed to the migration of the p-Chips. Like VIE tagging in San Marcos Salamanders (Phillips and Fries 2007), growth was unaffected by p-Chip tagging in Barton Springs, Comal Springs, and Texas Blind Salamanders in this study. However, we only examined growth at the conclusion of the study rather than at various points throughout the study, so we may have missed any variation in growth rates earlier in the study (e.g., Baras et al. 1999; Ruetz et al. 2006). Additionally, we did not examine any other metrics that might have been affected by tagging, such as behavior. Growth often affects the migration of subcutaneous tags in aquatic organisms (Linnane and Mercer 1998; Haddaway et al. 2011). For example, growth was shown to increase VIE breakage and deterioration in Eurycea spp. (Moon et al. 2022). However, we found no evidence of p-Chip migration with growth. Juvenile Texas Blind Salamanders had the highest growth rate but were also associated with 100% readability and tag retention. Growth might affect the migration of p-Chips used in individuals smaller than the ones we examined. More research is needed to evaluate the use of p-Chips in smaller (< 25 mm SVL) individuals of each of these species.
The results of this study show that P-Chips are suitable tags for small-bodied aquatic salamanders, especially for projects that require individual identification. However, other tags such as VIE tags might be more appropriate in projects where individual identification is not needed or short-term studies with few individuals, especially when costs must be reduced. The duration of effectiveness for subcutaneously injected p-Chips in aquatic organisms remains unknown. Longer-term monitoring is needed to determine the endpoint of p-Chip efficacy in long-lived aquatic organisms. Although no deterioration of p-Chip readability was observed over the eight months of this study, they may become more difficult to read with age. Additionally, more research is needed to determine the size limits of salamanders that can be tagged with p-Chips, particularly in the smaller salamander species in which we only tagged adults. Tagging juveniles could be beneficial for examining recruitment and growth rates, and for tracking individual metrics such as genetic, collection, and rearing information. Research comparing the efficacies of photo and p-Chip identification in these Species is needed. Photo identification is an effective method of the identification for some species at a low cost and might be preferable to, or used in conjunction with, p-Chips for some projects. Although we found no effect on growth from tagging with p-Chips, additional studies examining the effects of p-Chip tagging on behaviors such as swimming, hunting, and reproduction are recommended. Because survival and retention are often different in captive and wild settings (e.g., Musselman et al. 2017), studies examining the effects of p-Chips on organisms in the wild are needed to confirm the utility of this tagging method under wild conditions.
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Acknowledgments.—We thank Katie Bockrath, Justin Crow, and Braden West for support on this project. The Edwards Aquifer Authority provided project funding. This study was performed under the U.S. Fish and Wildlife Service permit TE676811-0 and the Texas Parks and Wildlife permit SPR-0622-090. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service. There 1s no conflict of interest declared in this article.
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Desiree Moore is a research biologist with the U.S. Fish and Wildlife Service and part of the Edwards Aquifer Refugia Program at the San Marcos Aquatic Resources Center (San Marcos, Texas, USA), conducting research to benefit federally listed aquifer species. Moore graduated with a Master of Science degree from Oklahoma State University (Norman, Oklahoma, USA) examining movement patterns and developing relationships between flow regime characteristics and occupancy probability of freshwater pelagic broadcast spawning minnows, with an emphasis on the federally threatened Arkansas River Shiner. Moore is continuing her research with federally listed aquatic organisms and specializes in tagging small-bodied aquatic animals.
Thomas Funk is a Biological Science Technician with the U.S. Fish and Wildlife Service at Inks Dam National Fish Hatchery in Burnet, Texas. He earned a Bachelor’s degree in Biology from the University of Georgia (Athens, Georgia, USA) before defending his Master’s thesis on differences in fish and decapod assemblages over natural and restored oyster reefs at Coastal Carolina University (Conway, South Carolina, USA). Currently, he works mostly in freshwater mussel propagation. His research is focused on optimizing the laboratory system rearing substrate and field grow-out system stocking density for propagated juvenile mussel survival and growth in Central Texas.
Madeleine Gillis earned a B.A. in Biology at St. Mary’s College of Maryland (St. Mary’s City, Maryland, USA). Her senior thesis was based on work she conducted as an intern studying great white sharks in South Africa. She attended Coastal Carolina University (Conway, South Carolina, USA) for graduate school and her Master’s thesis examined the effects of sublethal predation on marsh mussels and the predation preferences of blue crabs. After graduating, she stayed at CCU and taught introductory marine science lectures and labs before moving to Central Texas and interning at the San Marcos Aquatic Resources Center, mainly working on freshwater mussel propagation.
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Official journal website: amphibian-reptile-conservation.org
Amphibian & Reptile Conservation 18(1&2) [General Section]: 20-47 (e331).
The herpetofauna of Copia Nature Reserve, Vietnam
"Anh Van Pham, ??Truong Quang Nguyen, *4Tao Thien Nguyen, '**Minh Duc Le, ’®Thomas Ziegler, and *’Cuong Thien Tran
'Faculty of Environmental Sciences, University of Science, Vietnam National University, Hanoi, 334 Nguyen Trai Road, Hanoi 11400, VIETNAM Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi 10072, VIETNAM *Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi 10072, VIETNAM ‘Institute of Genome Research, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi 10072, VIETNAM SCentral Institute for Natural Resources and Environmental Studies, Vietnam National University, Hanoi, 19 Le Thanh Tong, Hanoi 11400, VIETNAM °Department of Herpetology, American Museum of Natural History, Central Park West at 79th Street, New York, New York 10024, USA 7AG Zoologischer Garten K6éln, Riehler StraBe 173, D-50735 Kéln, Germany *Institute of Zoology, University of Cologne, Ziilpicher Street 476, D-50674 Cologne, GERMANY
Abstract.—We report the results of a herpetofauna inventory of Copia Nature Reserve, Vietnam conducted between November 2012 and June 2020, comprising 48 species of amphibians and 67 species of reptiles, with 105 of the species recorded directly in this study. Four species, Gracixalus jinxiuensis, Dopasia ludovici, Hebius boulengeri, and Ovophis makazayazaya, represent new records for Son La Province, and 25 species are recorded for the first time from the Copia Nature Reserve, comprising 11 species of frogs (Xenophrys maosonensis, Microhyla mukhlesuri, Limnonectes bannaensis, Odorrana chapaensis, O. chloronota, O. jindongensis, O. graminea, O. nasica, Kurixalus bisacculus, Raorchestes parvulus, and Rhacophorus kio), two species of lizards (Calotes emma and Eutropis multifasciatus), and 12 species of snakes (Calamaria pavimentata, Dendrelaphis ngansonensis, Elaphe moellendorffi, E. taeniura, Euprepiophis mandarinus, Lycodon fasciatus, L. futsingensis, Oligodon fasciolatus, Sibynophis collaris, Psaammodynastes pulverulentus, Trimerodytes percarinatus, and Pareas carinatus). The herpetofauna of Copia Nature Reserve has a high level of conservation concern, including eight species listed in the Governmental Decree No. 84/2021/ND-CP, 17 species listed in the Vietnam Red Data Book (2007), 13 species listed in the IUCN Red List (2023), and eight species listed in the CITES Appendices (2023). In addition, we provide data on distribution, natural history, and figures for all the amphibian and reptile species in Copia Nature Reserve, Vietnam.
Keywords. Amphibians, biodiversity, distribution, natural history, new records, reptiles
Citation: Pham AV, Nguyen TQ, Nguyen TT, Le MD, Ziegler T, Tran CT. 2024. The herpetofauna of Copia Nature Reserve, Vietnam. Amphibian & Reptile Conservation 18(1&2): 20-47 (e331).
Copyright: © Pham, et al. 2024. This is an open access article distributed under the terms of the Creative Commons Attribution License [Attribution 4.0 International (CC BY 4.0): https://creativecommons.org/licenses/by/4.0/], which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The official and authorized publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation; official journal website: amphibian-reptile-conservation.org.
Accepted: 12 March 2024; Published: 5 September 2024
Introduction Copia Nature Reserve (NR). Further new records of reptiles and amphibians from this nature reserve were
The Copia Nature Reserve was established in documented by Pham et al. (2012, 2013, 2014b,c, November 2002 by the the People’s Committee 2015, 2017, 2018, 2019, 2020, 2022), and by Pham of Son La Province with an area of 11,996 ha. The 2d Nguyen (2018). Most recently, two new species nature reserve is situated in Thuan Chau District and Of amphibians and a new snake were described from is one of the five protected areas in Son La Province, C0P!aNR, namely /y/ototriton anguliceps Le, Nguyen, northern Vietnam (The People’s Committee of Son La Nishikawa, Nguyen, Pham, Matsui, Bernardes, and Province 2019). The topography of the nature reserve Nguyen, 2015 (Le et al., 2015a), Gracixalus truongi
is characterized by steep and mountainous terrain with Tran, Pham, Le, Nguyen, Ziegler, and Pham, 2023 elevations from 500 to 1,800 m asl. In terms of the (Tran et al. 2023), and Achalinus timi Ziegler, Nguyen,
herpetofaunal diversity, Le et al. (2009) provided the | Pham, Nguyen, Pham, Van Schingen, Nguyen, and Le, first list of 12 amphibian and 35 reptile species from 2019 (Ziegler et al. 2019). In addition, six new country
Correspondence. ‘phamanh@hus.edu.vn (AVP), tranthiencuong@hus.edu.vn (CTT)
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records of amphibians and reptiles have been reported from Copia NR, namely Boulenophrys daweimontis, Leptobrachella eos, L. minima, Leptobrachium masatakasatoi, Amolops vitreus, and Parafimbrios lao (Pham et al. 2014a, 2016; Le et al. 2015b; Nguyen et al. 2015). As a result of our ongoing research over the past ten years, we herein provide an updated list, with new data on the distribution and natural history of amphibians and reptiles from Copia NR.
Materials and Methods
Sixteen field surveys were conducted at six sites in Copia NR, Son La Province, Vietnam, including a total of 95 days. Survey sites were set up in the forests near Huoi Pu and Hua Ty A villages, Chieng Bom Commune; near Nong Vai, Pha Khuo ng, and Co Ma villages, Co Ma Commune; and near Long He Village, Long He Commune by A.V. Pham, T.Q. Nguyen, T.T. Nguyen, N.B. Sung, H.V. Tu, T.V. Nguyen (Fig. 1 and Table 1).
The typical habitats at the study sites were undisturbed evergreen forest, secondary forest, and agricultural areas (Fig. 2). The geographical coordinates (WGS84) of all observations were recorded using a Garmin GPSMAP 62s. Specimens were collected by hand between 0800-2300 h. After they were photographed in life, specimens were identified to the species level, measured, sexed, and released at the collection site. For voucher specimens, a few individuals were anesthetized and euthanized in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmon 2002), fixed in 80% ethanol, and then transferred to 70% ethanol for permanent storage.
Some road-killed specimens were also collected for morphological examination. These specimens were subsequently deposited in the collection of the University of Science, Vietnam National University (VNU), Hanoi, Vietnam.
For taxonomic identification, we referred to the descriptions in Bain et al. (2003), Boulenger (1893), Bourret (1942), Inger et al. (1999), Fei et al. (2012), Hecht et al. (2013), Smith (1935, 1943), and Taylor (1962). For species names, we followed Frost (2023) for amphibians and Uetz et al. (2023) for reptiles.
Conservation status of amphibian and reptile species followed the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES 2023), the Red List of the International Union for Conservation of Nature (IUCN 2023), the Vietnam Red Data Book (Dang et al. 2007), and the Governmental Decree No 84/2021/ND-CP, issued on 22 September 2021 by the Government of Vietnam, on the management of endangered species of wild flora and fauna.
Results
A total of 115 species belonging to 78 genera and 27 families were recorded from Copia NR, comprising 48 species of amphibians (28 genera, seven families) and 67 species of reptiles (50 genera, 20 families) (Table 2). Remarkably, one species of frog, one species of lizard and two species of snakes are reported for the first time from Son La Province; and 25 additional species are documented for the first time from Copia NR, comprising 11 species of anurans, two species of lizards, and 12 species of snakes.
Table 1. Information on the survey sites in Copia Nature Reserve, Vietnam.
No Site Survey dates Latitude Longitude eae Forest near Huoi Pu Village, Chieng
1 Bom Commune, Thuan Chau eee ye 21°23.110°N 103°38"522”E 860
is 17 to 24 June 2016
District Forest near Hua Ty A Village, 20 to 25 November 2012;
ih Chieng Bom Commune, Thuan 20 to 25 March 2013; 21°21.210’N ~—-103°35.566’E 960 Chau District 2 to 13 September 2016 Forest near Nong Vai Village, Co Ma 11 to 19 June 2013; é ; : ;
3 Commune, Thuan Chau District 16 to 22 October 2016 21°18.589°N —103°33.250°E 1,450 Forest near Pha Khuong Village, Co - : a ,
4 Ma Conimiune Thuan Chau District 4 to 15 July 2013 21°21.426’N ~——-103°31.230’E 1,260 Forest near Co Ma Village, Co Ma 6 ; a :
> Commune, Thuan Chau: District 19 to 24 May 2015 21°21.469"N ——-103°30.380’E 1,090 Forest near Long He Village, Long 22 to 31 August 2014; 4 , a :
s He Commune, Thuan Chau District 18 to 29 April 2020 21°24.130°N — 103°29.238°E 1,010
Amphib. Reptile Conserv.
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103° 31' 48" B 103° 36' 00" D 103° 40' 12"
Hoang Sa Arch (Vietnam)
.
Truong Sa Arch (Vietnam)
Ratio 1:25.000 | 103° 31'48"D 103° 40 12"
Fig. 1. Survey sites in Copia Nature Reserve, Son La Province, Vietnam. 1. Huoi Pu Village, Chieng Bom Commune; 2. Hua Ty A Village, Chieng Bom Commune; 3. Nong Vai Village, Co Ma Commune; 4. Pha Khuong Village, Co Ma Commune; 5. Co Ma Village, Co Ma Commune; and 6. Long He Village, Long He Commune.
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Fig. 2. Habitat types in Copia Nature Reserve, Vietnam. (A, B, C) Evergreen forest, (D) Disturbed secondary forest, and (E, F) Agricultural areas.
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The herpetofauna of Copia Nature Reserve, Vietnam
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The herpetofauna of Copia Nature Reserve, Vietnam
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‘(EZOT) ‘Te 19 UOANSN = BI “(ZZOT) ‘Te 19 Wed = LT (6107) ‘Te 19 JO[S9IZ = 91 (6107) ‘Te 19 WeYd = ST (8107) ‘Te 0 WeYyd = FI (8107) uadnsN pue weyd = €1 (L107) Te 10 Weyd = ZI (9107) [e190 WeYyd = TT (S107) Te 19 UOANSN = OT “(GSTOZ) 1219 9T=6 (eSTOZ) 1219 OT = 8 “(SI10Z) Te 10 Wey = L “(Op10Z) ‘Te 10 WIRY = 9 (qrI0Z) [e319 Wey =¢ (eP[OZ) Je 10 WeUd = p (E107) Je 10 WeUd = € ‘(ZI0Z) Je 19 WeUd =Z ‘(600Z) [e 19 YT = | :UUIN[OS P1091 SNOTADIg 9Y} UI SUOTIEIID “(asodind yerssOUIWI0d JOF asn pue uoneo[dxs pouty) qI] dno1pH = q]] :WeujsiA JO JUSWIUIDAOD ay} Aq ‘[ZOZ Joquiajdas ZZ UO palep G@D-CN/IZOTZ/P8 ‘ON 90190] [VJUSWIUIDAOD d= p8 ON 9901990q] “]] pue | xIpuoddy = II I (€707 SALIO) soorpuodde Sq LID = SALIO ‘pousyeosy , LON /STY JOMOT = LN “O[GeIOUINA = (A ‘pososuepug = Nq ‘pososuepug ATPONWD = YO (€ZOT NOND sotoadg pouayeosy I, JO ISI] POY NONI SUL = NONI:
‘(LOOT ‘Je 19 Bue) WeUIIIA JO joo, ve poy ‘svore [eINyNOLISV = ¢ pure ‘JsaIoJ ATepuodas poqiNIsIq = TZ ‘JSOIOJ USSIBIOAY = | ‘JeUGeH ‘| 2[QeI, Ul UMOYS sv 9 0} [| WIOLJ SOUS SULIOQUINN ‘O11 SYN PIdOD JO} P1099 MOU = ,, “DOUTAOI_ BT UOS JO} P1ONII MOU = yy WRIA YN bIdoD Wo papsooal sordeds ayda1 puv ueIqiydure Jo jsI'T ‘panuljyuo, Z I[quy,
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x(P98[ JoyUNyH) snpojolsspf uoposyoO
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(8p 107) Te 19 wed = f pue uoneyo[dxe poyuny) qI] dno1p = q]] :WeujsiA JO JUSWIUIDAODH ay} Aq “[ZOZ Joquiajdas ZZ UO palep G@D-CN/IZOTZ/P8 ‘ON 90190] [VJUSWIUIDAOD SU] = 78 ON 991990q] “]] pue | xIpuoddy = II I (€707 SALID) soorpuodde Sq LID = SALIO ‘pousyeosyL, LON/STY JOMOT = LN “O[GesJOUNA = (A “pososuepug = Nq ‘pososuepug ATBoNWD = YO (€Z7OT NOND sotoadg pouayeosy I, JO IST POW NONI ULL = NONI “(LOOT Te 19 Sued) weujor, JO yoo vjeq poy ‘svore [eININILIsVy = ¢ pure ‘Jsa1oJ AJepuOdaS poqiN}sIq = J ‘JSOIOJ USSIBIDAY = | JeUQeH *] quel, Ul UMOYS se 9g 0}
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| WOIJ SozS SULIOQUINN :}1S “YN PIdOD JO} P1OIII MOU = x, SQOUIAOIY BT UOS JO} P1OIOI MOU = yy WRUIIIA YN eIdoD Wo papsosel sardeds apndos pue ueiqrydure Jo js] ‘ponunyues Z 3qeL
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oupyeuLigpoudXy
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| WOIJ SopS SULIOQUINN :3}1S SYN PIdOD JO} p1OIII MOU = y, SDOUIAOIY BT UOS JO} P1OIOI MOU = yy SWLUIIIA YN eIdoDd wo papsodel satdeds apndos pue ueiqrydure jo jsI’] ‘panuyuos Z 3qeL
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a dq (Z88] ‘JayjuaNy) vssaidu viinounpy
6107 ‘OJ pue ‘uadAnsN 91 I £ ‘UdSUIYSS uUvA ‘Wey, ‘UdANSN “wey, “UdANSNY “IO[SOIzZ 11 snuljOYyoy
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Amphibia Anura Bufonidae
Duttaphrynus melanostictus (Schneider, 1799) (Fig. 3A): Individuals were observed at night on the ground in croplands, gardens, and road edges near residential areas and disturbed secondary forest.
Megophryidae
Boulenophrys daweimontis (Rao and Yang, 1997) (Fig. 3B): One specimen was found at night on the ground, near a stream in evergreen forest.
Boulenophrys_ palpebralespinosa (Bourret, 1937) (Fig. 3C): Two specimens were found at night on leaves, at ca. 30-90 cm above the ground near a stream, and many other individuals were observed along streams at night in evergreen forest.
Boulenophrys cf. parva (Boulenger, 1893) (Fig. 3D): Two specimens were found at night on the ground, near a stream in evergreen forest. Boulenophrys parva seems to be restricted in Myanmar and records of this species in northern Vietnam should be assigned to other named and unnamed species (Manhony et al. 2020).
Leptobrachella eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler, and Dubois, 2011) (Fig. 3E): Two Specimens were found at night on the ground near streams and many other individuals were observed at night on the ground along streams in evergreen forest.
Leptobrachella minima (Taylor, 1962) (Fig. 3F): Two Specimens were found at night on the ground near a stream in evergreen forest.
Leptobrachella ventripunctata (Fei, Ye, and Li, 1990) (Fig. 3G): Two specimens were found on the ground near streams and many other individuals were observed along streams at night in evergreen forest.
Leptobrachium masatakasatoi Matsui, 2013 (Fig. 3H): Three specimens were found at night on the ground near a stream in evergreen forest.
Ophryophryne pachyproctus Kou, 1985 (Fig. 31): Two specimens and many others were found at night on leaves, at ca. 50-120 cm above the ground near streams, and many other individuals were observed on leaves along streams at night in evergreen forest.
Xenophrys maosonensis (Bourret, 1937) (Fig. 3J): Two specimens were found at night on the ground near a stream, and many other individuals were observed along streams at night in evergreen forest. This is a new record for Copia NR.
Amphib. Reptile Conserv.
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Microhylidae
Kaloula pulchra Gray, 1831 (Fig. 3K): Two individuals were observed at night on the ground in a garden near a pond.
Microhyla butleri Boulenger, 1900 (Fig. 3L): Two speci- mens were found at night on the ground in meadowlands, and other individuals were observed at night in croplands and small puddle edges near the rice fields and forest edges.
Microhyla heymonsi Vogt, 1911 (Fig. 3M): Two specimens were found at night on the ground in croplands, and other individuals were observed at night on the ground in croplands in meadowlands, and along forest trails near forest edges and inside the forest.
Microhyla mukhlesuri Hasan, Islam, Kuramoto, Kurabayashi, and Sumida, 2014 (Fig. 3N): Two specimens were found at night on the ground near small puddle edges near a road, and other individuals were observed at night on the ground near small puddle edges in rice fields, meadowlands, and croplands. This is a new record for Copia NR.
Microhyla_ pulchra_ (Hallowell, 1861) (Fig. 30): Three specimens were found at night on the ground in meadowlands near rice fields, and other individuals were observed at night on the ground around small puddle edges and in meadowlands near rice fields.
Micryletta menglienica (Yang and Su, 1980) (Fig. 3P): Two specimens were found at night on the ground in meadowlands near limestone mountains. This is a new record for Copia NR.
Dicroglossidae
Fejervarya limnocharis (Gravenhorst, 1829) (Fig. 3Q): Many individuals were observed at night on the ground, as well as in meadowlands near rice fields, croplands, and small puddles at road edges.
Hoplobatrachus chinensis (Osbeck, 1765) (Fig. 3R): Five individuals were observed at night on the ground, and at pond edges and rice field edges.
Limnonectes bannaensis Ye, Fei, Xie, and Jiang, 2007 (Fig. 4A): Two specimens were found at night on the ground near a stream, and many other individuals were observed at night on the ground near streams or water edges of streams in evergreen forest. This is a new record for Copia NR.
Nanorana aenea (Smith, 1922) (Fig. 4B): Two specimens were found at night on the ground near a stream, and many other individuals were observed at night on the ground near streams in evergreen forest.
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Fig. 3. Amphibian species recorded in Copia Nature Reserve, Vietnam. (A) Duttaphrynus melanostictus, (B) Boulenophrys daweimontis, (C—D) B. Palpebralespinosa, (E) Leptobrachella eos, (F) L. minima, (G) L. ventripunctata, (H) Leptobrachium masatakasatoi, (1) Ophryophryne pachyproctus, (J) Xenophrys maosonensis, (K.) Kaloula pulchra, (L) Microhyla butleri, (M) M. heymonsi, (N) M. mukhlesuri, (0) M. pulchra, (P) Micryletta menglienica, (Q) Fejervarya limnocharis, and (R) Hoplobatrachus
chinensis.
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Fig. 4. Amphibian species recorded in Copia Nature Reserve, Vietnam. (A) Limnonectes bannaensis, (B) Nanorana aenea, (C) Quasipaa verrucospinosa, (D) Amolops cf. compotrix, (E) A. vitreus, (F) Odorrana chapaensis, (G) O. chloronota, (H) O. graminea, (1) O. jingdongensis, (J) O. nasica, (K) Sylvirana guentheri, (L) S. nigrovittata, (M) Chirixalus doriae, (N) Gracixalus jinxiuensis, (O) G. quangi, (P) G. truongi, (Q) Kurixalus bisacculus, and (R) Polypedates megacephalus.
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Quasipaa verrucospinosa (Bourret, 1937) (Fig. 4C): Individuals were observed at night on rocks in streams or near waterfalls in evergreen forest.
Ranidae
Amolops cf. compotrix (Bain, Stuart, and Orlov, 2006) (Fig. 4D): Two specimens were found at night on tree branches near streams. The surrounding habitat was evergreen forest, composed of small hardwoods, liane, and shrub. This is a new record for Copia NR.
Amolops vitreus (Bain, Stuart, and Orlov, 2006) (Fig. 4E): Two specimens were found at night on tree branches near a stream, and six other individuals were observed at night on tree branches near streams in evergreen forest.
Odorrana chapaensis (Bourret, 1937) (Fig. 4F): Two specimens were found at night on a rock near a waterfall in evergreen forest. This is a new record for Copia NR.
Odorrana chloronota (Gunther, 1876) (Fig. 4G): Two specimens were found at night on a rock near a stream, and many other individuals were observed at night on tree branches or on rocks near streams in evergreen forest. This is anew record for Copia NR.
Odorrana graminea Boulenger, 1900 (Fig. 4H): One specimen was found at night on a rock near a waterfall in evergreen forest. This is a new record for Copia NR.
Odorrana jingdongensis Fei, Ye, and Li, 2001 (Fig. 41): Two specimens were found at night on a tree branch near a stream, and many other individuals were observed at night on tree branches or on rocks at ca. 0-80 cm above the ground near streams in evergreen forest. This is a new record for Copia NR.
Odorrana nasica (Boulenger, 1903) (Fig. 4J): Two Specimens were found at night on a tree branch near a waterfall, and many other individuals were observed at night on tree branches or on rocks at ca. 0-90 cm above the ground near waterfalls in evergreen forest. This is a new record for Copia NR.
Sylvirana guentheri (Boulenger, 1882) (Fig. 4K): Individuals were observed at night on the ground near pond edges and streams. The surrounding habitat was rice fields.
Sylvirana_ nigrovittata (Blyth, 1856) (Fig. 4L): Two specimens were found at night on the ground near a stream, and other individuals were observed at night on the ground, and on stones near streams or at the water edge in streams. The surrounding habitat was evergreen forest. The call concerts of this species were regularly heard in the evening.
Amphib. Reptile Conserv.
Rhacophoridae
Chirixalus doriae Boulenger, 1893 (Fig. 4M): One specimen was found at night on a tree branch near a puddle, and five individuals were observed at night while sitting on leaves near puddles at ca. 30-80 cm above the ground. The surrounding habitat was mixed evergreen forest of small hardwoods, bamboo, and shrubs.
Gracixalus jinxiuensis (Hu, 1978) (Fig. 4N): Two specimens were found at night while sitting on leaves near a stream at ca. 1-2 m above the ground. The surrounding habitat was mixed evergreen forest of small hardwoods, bamboo, and shrubs. This is a new record for Son La Province.
Gracixalus quangi Rowley, Dau, Nguyen, Cao, and Nguyen, 2011 (Fig. 40): Two specimens were found at night on leaves near a stream, and other individuals were observed at night while sitting on leaves near streams at ca. 0.8—1.5 m above the ground. The surrounding habitat was mixed evergreen forest of small hardwoods, bamboo, and shrubs.
Gracixalus truongi Tran, Pham, Le, Nguyen, Ziegler, and Pham, 2023 (Fig. 4P): Two specimens were found at night while sitting on leaves at ca. 1-1.5 m above the ground. The surrounding habitat was mixed evergreen forest of small hardwoods, bamboo, and shrubs in limestone mountains.
Kurixalus bisacculus (Taylor, 1962) (Fig. 4Q): Two Specimens were found at night on the tree branches near puddles, and other individuals were observed at night while sitting on leaves or branches near streams or puddles at ca. 0.2-3.0 m above the ground. The surrounding habitat was cultivated land and mixed evergreen forest of small hardwoods, bamboo, and shrubs. This is a new record for Copia NR.
Polypedates megacephalus Hallowell, 1861 (Fig. 4R): Two specimens were found at night on the tree branches near a stream, and other individuals were observed at night while sitting on leaves or branches near streams, puddles, and ponds at ca. 0.3—2.5 m above the ground. The surrounding habitat was cultivated land and mixed evergreen forest of small hardwoods, bamboo, and shrubs. This is a new record for Copia NR.
Raorchestes parvulus (Boulenger, 1893) (Fig. 5A): Two Specimens were found at night sitting on leaves near streams, and other individuals were observed on the same perch sites at ca. 1.54.0 m above the ground in evergreen forest. This is a new record for Copia NR.
Rhacophorus kio Ohler and Delorme, 2006 (Fig. 5B):
Individuals were observed at night sitting on leaves near puddles at ca. 1-3 m above the ground in evergreen forest.
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Fig. 5. Amphibian species recorded in Copia Nature Reserve, Vietnam. (A) Raorchestes parvulus, (B) Rhacophorus kio, (C) R. rhodopus, (D) Theloderma albopunctatum, (E) T: bicolor, (F) T: corticale, (G) T. gordoni, (H) Zhangixalus dorsoviridis, (1) Z. feae, and (J) Zylototriton anguliceps.
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Rhacophorus rhodopus Liu and Hu, 1960 (Fig. 5C): One specimen was found at night sitting on leaves near puddles, and other individuals were observed at night while sitting on the same perch sites at ca. 2—3 m above the ground in evergreen forest.
Theloderma albopunctatum (Liu and Hu, 1962) (Fig. 5D): One specimen was found at night on edge of the waterhole and six individuals were observed at night at the same perch sites in evergreen forest.
Theloderma bicolor (Bourret, 1937) (Fig. 5E): One specimen was found at night on edge of a water filled tree hole in evergreen forest.
Theloderma corticale (Boulenger, 1903) (Fig. 5F): One specimen was found at night on edge of a water filled tree hole in evergreen forest.
Theloderma gordoni Taylor, 1962 (Fig. 5G): Two specimens were found at night on edge of a water filled tree hole in evergreen forest.
Zhangixalus dorsoviridis (Bourret, 1937) (Fig. 5H): Two specimens were found at night on the tree branches near a stream in evergreen forest.
Zhangixalus feae (Boulenger, 1893) (Fig. 5I): Individuals were observed at night while sitting on tree branches or leaves near streams at 1-4 m above the ground in evergreen forest.
Caudata Salamandridae
Tylototriton anguliceps Le, Nguyen, Nishikawa, Nguyen, Pham, Matsui, Bernardes, and Nguyen, 2015 (Fig. 5J): Individuals were observed during the daytime underneath the carpet of fallen leaves near small streams in evergreen forest.
Reptilia Squamata Agamidae
Acanthosaura lepidogaster (Cuvier, 1829) (Fig. 6A): Individuals were observed while sitting on trees at ca. 1-2 m above the ground or while crossing a forest path in evergreen forest.
Calotes emma Gray, 1845 (Fig. 6B): One specimen was found in the morning on a forest path in evergreen forest. This is a new record for Copia NR.
Calotes versicolor (Daudin, 1802) (Fig. 6C):
Individuals were observed during the daytime near cultivated lands or bushes in gardens.
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Draco maculatus (Gray, 1845) (Fig. 6D): A road- killed individual was found on Road 108. The surrounding habitat was mixed evergreen forest of hardwoods and shrubs.
Pseudocalotes brevipes (Werner, 1904) (Fig. 6E): One specimen was found at night on a tree branch, and six individuals were observed at night while sitting on the same perch sites at ca. 1-2 m above the ground in evergreen forest.
Gekkonidae
Gekko reevesii (Gray, 1831) (Fig. 6F): Individuals were observed at night on limestone cliffs or large trees at ca. 1-6 m above the ground in evergreen forest.
Hemidactylus frenatus Duméril and Bibron, 1836 (Fig. 6G): Individuals were observed at night on a wall near a light bulb in a residential area.
Hemidactylus garnotii Duméril and Bibron, 1836 (Fig. 6H): Two specimens were found and other individuals were observed at night on limestone karst outcrops at 1-3 m above the ground in secondary forest.
Scincidae
Eutropis longicaudatus (Hallowell, 1857) (Fig. 61): Individuals were observed during the daytime on the ground at road edges, and on shrubs near cultivated land.
Eutropis multifasciatus (Kuhl, 1820) (Fig. 6J): Individuals were observed during the daytime on the ground at road edges, and on shrubs near cultivated land. This is a new record for Copia NR.
Plestiodon cf. tamdaoensis (Bourret, 1937) (Fig. 6K): One specimen was found during the daytime on the ground at road edges near cultivated land. This is a new record for Copia NR.
Scincella devorator Darevsky, Orlov, and Ho, 2004 (Fig. 6L): One specimen was found in the afternoon on a forest path in evergreen forest.
Sphenomorphus indicus (Gray, 1853) (Fig. 6M): One specimen was found in the afternoon on a forest path and other individuals observed on the ground in evergreen forest.
Tropidophorus baviensis Bourret, 1939 (Fig. 6N).:
Two specimens were found under a carpet of fallen leaves, at a forest edge near evergreen forest.
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Fig. 6. Lizard species recorded in Copia Nature Reserve, Vietnam. (A) Acanthosaura lepidogaster, (B) Calotes emma, (C) C. versicolor, (D) Draco maculatus, (E) Pseudocalotes brevipes, (F) Gekko reevesii, (G) Hemidactylus frenatus, (H) H. garnotii, (1) Eutropis longicaudatus, (J) E. multifasciatus, (K) Plestiodon cf. tamdaoensis, (L) Scincella devorator, (M) Sphenomorphus indicus, (N) Tropidophorus baviensis, (0) Dopasia harti, and (P) D. ludovici.
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Dopasia harti (Boulenger, 1899) (Fig. 60): One specimen was found during the daytime on the ground at road edges near evergreen forest.
Dopasia ludovici (Mocquard, 1905) (Fig. 6P): Two specimens were found during the daytime on the ground at road edges near evergreen forest. This is a new record for Son La Province.
Typhlopidae
Indotyphlops braminus (Daudin, 1803) (Fig. 7A): Two specimens were found in the afternoon underneath a rotten plank near the forest edge.
Xenopeltidae
Xenopeltis unicolor Reinwardt, 1827 (Fig. 7B): Two individuals were observed at night on the ground near the forest edge.
Colubridae
Ahaetulla prasina (Boie, 1827) (Fig. 7C): Individuals were found at night on tree branches at ca. 1.0-2.5 m above the ground in secondary forest or on a fence near agricultural areas.
Boiga multomaculata (Boie, 1827) (Fig. 7D): Three individuals were observed at night on tree branches at ca. 1—3 m above the ground in evergreen forest.
Calamaria pavimentata Dumeéril, Bibron, and Dumeéril, 1854 (Fig. 7E): One specimen was found at night on the ground while moving across a forest trail near evergreen forest. This is a new record for Copia NR.
Coelognathus radiatus (Boie, 1827) (Fig. 7F): Two individuals were observed in the afternoon while moving across a road near agricultural areas.
Dendrelaphis ngansonensis (Bourret, 1935) (Fig. 7G): One specimen was found during the daytime on the ground on a road edge near the evergreen forest. This is a new record for Copia NR.
Dendrelaphis pictus (Gmelin, 1789) (Fig. 7H): Two Specimens were found during the daytime on tree branches at road edges near evergreen forest.
Elaphe moellendorffi (Boettger, 1886) (Fig. 71): One individual was observed at 1805 h near the entrance of a cave at a forest edge. This is a new record for Copia NR.
Elaphe taeniura (Cope, 1861) (Fig. 7J): Two individuals
were observed in the afternoon at the entrance of a cave near a forest edge and agricultural area. This is a new
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record for Copia NR.
Euprepiophis mandarinus (Cantor, 1842) (Fig. 7K): One individual was observed at night on the ground near a stream in evergreen forest. This 1s a new record for Copia NR.
Gonyosoma coeruleum Liu, Hou, Lwin, Wang, and Rao, 2021 (Fig. 7L): One individual was observed at night on a tree branch near a stream in evergreen forest. Previous records of Gonyosoma prasinum in Copia NR by Pham et al. (2014) should be re-identified as B. coeruleum after Liu et al. (2021).
Gonyosoma frenatum (Gray, 1853) (Fig. 7M): A road- killed specimen was found in the morning on a road near evergreen forest.
Liopeltis frenata (Gunther, 1858) (Fig. 7N): A road- killed specimen was found in the morning on a road near evergreen forest.
Lycodon fasciatus (Anderson, 1879) (Fig. 70): One specimen was found at night on the ground while catching prey near a road, and five other individuals were observed at night on the ground near streams or forest trails in evergreen forest. This is a new record for Copia NR.
Lycodon futsingensis (Pope, 1928) (Fig. 7P): One specimen was found at night on the ground near a stream in evergreen forest. This is a new record for Copia NR.
Oligodon catenatus (Blyth, 1854) (Fig. 7Q): One specimen was found at night on the ground near a forest trail in evergreen forest, and a road-killed specimen was found on the road.
Oligodon fasciolatus (Gunther, 1864) (Fig. 7R): One specimen was found at night on the ground in evergreen forest. This is a new record for Copia NR.
Oreocryptophis porphyraceus (Cantor, 1839) (Fig. 8A): One individual was observed at night on the ground while moving near a forest trail in evergreen forest.
Ptyas korros (Schlegel, 1837) (Fig. 8B): Two individuals were observed during the daytime on the ground at the roadside near a forest edge.
Ptyas multicincta (Roux, 1907) (Fig. 8C): One individual was observed at night on a tree branch in evergreen forest.
Sibynophis collaris (Gray, 1853) (Fig. 8D): One specimen was found during the daytime on the ground while moving across the road. This is a new record for Copia NR.
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Fig. 7. Snake species recorded in Copia Nature Reserve, Vietnam. (A) /ndotyphlops braminus, (B) Xenopeltis unicolor, (C) Ahaetulla prasina, (D) Boiga multomaculata, (E) Calamaria pavimentata, (F) Coelognathus radiatus, (G) Dendrelaphis ngansonensis, (H) D. pictus, (1) Elaphe moel- lendorffi, (J) Elaphe taeniura, (K) Euprepiophis mandarinus, (L) Gonyosoma coeruleum, (M) G. frenatum, (N) Liopeltis frenata, (O) Lycodon
fasciatus, (P) L. futsingensis, (Q) Oligodon catenatus, and (R) O. fasciolatus.
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Elapidae
Bungarus fasciatus (Schneider, 1801) (Fig. 8E): One individual was observed at night on the ground while moving across the road near secondary forest.
Bungarus wanghaotingi Pope, 1928 (Fig. 8F): Two individuals were observed near a stream in secondary forest. Previous records of B. multicinctus in Copia NR by Le et al. (2009) and in Vietnam by Nguyen et al. (2009) should be re-identified as B. wanghaotingi after Chen et al. (2021). Chen et al. (2021) stated that previous records of B. multicinctus in Vietnam and southern China should be re-identified as B. wanghaotingi. However, these species are difficult to distinguish morphologically. Bungarus wanghaotingi differs from B. multicinctus by having fewer light cross bands on the body and tail (20-31 and 7-11 versus 31—40 on the body and 9-17 on the tail in B. multicinctus, respectively). The specimens from Copia NR have 25—26 and seven light cross bands on the body and tail, respectively.
Naja atra Cantor, 1842 (Fig. 8G): Two individuals were observed during the daytime on the ground near bamboo bush in secondary forest.
Sinomicrurus macclellandi (Reinhardt, 1844) (Fig. 8H): One individual was observed at night on the ground while moving near a forest trail in evergreen forest.
Lamprophiidae
Psammodynastes pulverulentus (Boie, 1827) (Fig. 81): One individual was observed at night on a tree branch in limestone forest. This is anew record for Copia NR.
Natricidae
Fowlea flavipunctatus (Hallwell, 1861) (Fig. 8J): An individual was observed in the afternoon near a pond. The surrounding habitat was secondary forest.
Hebius boulengeri (Gressitt, 1937) (Fig. 8K): Two specimens were found at night on the ground near a stream in evergreen forest. This is a new record for Copia NR.
Hebius chapaensis (Bourret, 1934) (Fig. 8L): One specimen was found at night in a stream in evergreen forest.
Rhabdophis subminiatus (Schlegel, 1837) (Fig. 8M): Three individuals were observed during the daytime on grass near the roadside in an agricultural cultivated area.
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Trimerodytes percarinatus (Boulenger, 1899) (Fig. 8N): Two individuals were observed at night in streams in evergreen forest. This is a new record for Copia NR.
Pareatidae
Pareas carinatus (Boie, 1828) (Fig. 80): One specimen was found at night while sitting on a tree branch in the secondary forest. This is a new record for Copia NR.
Pareas hamptoni (Boulenger, 1905) (Fig. 8P): One specimen was found at night while sitting on a tree branch, and many other individuals were observed at night on tree branches at ca. 0.6—1.5 m above the ground in evergreen forest.
Pareas margaritophorus (Jan, 1866) (Fig. 8Q): Two specimens were found at night while sitting on tree branches at ca. 0.6—1.0 m in evergreen forest.
Pseudoxenodontidae
Pseudoxenodon macrops (Blyth, 1854) (Fig. 8R): Two specimens were found in the morning while moving across a forest trail in evergreen forest.
Viperidae
Ovophis makazayazaya (Takahashi, 1922) (Fig. 9A): Two specimens were found at night while moving across a forest trail in evergreen forest. This is a new record for Son La Province.
Ovophis monticola (Gunther, 1864) (Fig. 9B): Two specimens were found at night on the ground near streams in evergreen forest.
Trimeresurus albolabris Gray, 1842 (Fig. 9C).: Individuals were observed during the daytime on tree branches at ca. 0.5-1.2 m above the ground near secondary forest and cultivated agricultural areas.
Testudines Platysternidae
Platysternon megacephalum Gray, 1831 (Fig. 9D): Two individuals were observed at night under a rock in a stream in evergreen forest.
Testudinidae
Manouria impressa (Guenther, 1882) (Fig. 9E): One
individual was observed during the daytime in a house of the local people.
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Fig. 8. Snake and turtle species recorded in Copia Nature Reserve, Vietnam. (A) Oreocryptophis porphyraceus, (B) Ptyas korros, (C) P. multicincta, (D) Sibynophis collaris, (E) Bungarus fasciatus, (F) B. wanghaotingi, (G) Naja atra, (H) Sinomicrurus macclellandi, (1), Psammodynastes pulverulentus, (J) Fowlea flavipunctatus, (K) Hebius boulengeri, (L) Hebius chapaensis, (M) Rhabdophis subminiatus, (N) Trimerodytes
percarinatus, (O) Pareas carinatus, (P) P. hamptoni, (Q) P. margaritophorus, and (R) Pseudoxenodon macrops.
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Fig. 9. Snake and turtle species recorded in Copia Nature Reserve, Vietnam. (A) Ovophis makazayazaya, (B) O. monticola, (C) Trimeresurus albolabris, (D) Platysternon megacephalum, and (E) Manouria impressa.
Discussion
Our new findings bring the number of amphibian and reptile species in Copia NR to 115, comprising 48 amphibian and 67 reptile species, and 25 of the species are new records for Copia NR while four are new records for Son La Province.
Among the six survey sites, the forest sites near Nong Vai and Hua Ty villages had the highest level of species richness, with 65 recorded species; followed by Huoi Pu forest with 52 species; Co Ma site with 37 species; Long He site with 36 species; and Pha Khuong site with 32 species (Table 2). The forest sites near Nong Vai and Hua Ty are in the core zone of the Copia NR with a large area of evergreen forest (>2,000 ha) and the habitat quality is relatively good. This explains why the number of recorded species is higher than those of other sites.
In terms of habitat preference, most of the amphibians and reptiles inhabit the evergreen forest (63 species, or 60% of the total recorded species), followed by disturbed secondary forest with 34 recorded species (32.38%), and agricultural areas with 24 recorded species (22.85%) (Table 2).
Concerning its herpetofaunal conservation status, Copia NR harbors many threatened species, including 17 species listed in the Red Data Book of Vietnam (Dang et al. 2007), with three species categorized as CR (Boulenophrys_ palpebralespinosa, Python bivittatus, and Ophiophagus hannah), 10 species as EN (Rhacophorus kio, Theloderma corticale, Zhangixalus feae, Varanus salvator, Coelognathus radiatus, Ptyas
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korros, Ptyas mucosa, Bungarus fasciatus, Naja atra, and Platysternon megacephalum), and four species as VU (Elaphe moellendorffi, Euprepiophis mandarinus, Oreocryptophis porphyraceus, and Manouriaimpressa). In addition, 13 species are listed in the IUCN Red List (IUCN 2023), with two species categorized as CR (P. megacephalum and Mauremys sinensis), one species as EN (M. impressa), eight species as VU (Amolops vitreus, Odorrana graminea, Odorrana jingdongensis, Zhangixalus dorsoviridis, E. moellendorffi, Elaphe taeniura, N. atra, and O. hannah), and two species as NT (P. molurus and P. korros). Eight species are listed in the Vietnam Governmental Decree No. 84/2021/ND- CP (2021), with two species included in Group IB (O. hannah and P. megacephalum) and six species in Group IIB (7vlototriton anguliceps, V. salvator, P. molurus, P. mucosus, N. atra, and M. impressa). Furthermore, eight species are listed in the CITES appendices, with one species included in Appendix I (P. megacephalum) and seven species in Appendix II (7 anguliceps, V. salvator, P. molurus, P. mucosus, N. atra, O. hannah, and M. impressa) (Table 2).
Acknowledgements.—We are grateful to the directorates of Forest Protection Department of Son La Province and Copia Nature Reserve for their support of our field work and issuing the relevant permits (permit No. 22/GT issued on 7 June 2012). We thank NB Sung (Son Ma District, Son La Province), TV Nguyen (Ninh Binh Province), QT Bui (Son La Province), HV Tu (Bac Giang Province), TQL Hoang (Son La Province), and MA Sung (Thuan Chau District, Son La Province) for their assistance in the field.
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Anh Van Pham is an Associate Professor of Biology and a Senior Lecturer at the Faculty of Environmental Sciences, University of Science, Vietnam National University, Hanoi. His research focuses on the taxonomy and conservation of amphibians and reptiles in Vietnam. He has published one book and 80 articles, mainly dealing with the herpetodiversity of Vietnam.
Truong Quang Nguyen is a Senior Researcher at the Institute of Ecology and Biological Resources and a Professor of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology in Hanoi. His research interests include the systematics, ecology, phylogeny, and conservation of reptiles and amphibians in Southeast Asia. He is the co-author of 20 books and more than 350 articles related to biodiversity research and conservation in Southeast Asia.
Tao Thien Nguyen is a Senior Researcher at the Institute of Genome Research, Vietnam Academy of Science and Technology in Hanoi. His research interests include the taxonomy and evolution of amphibians and reptiles. Tao is also working on the conservation and sustainable use of reptiles and amphibians on Vietnam. He has published several books and more than 150 academic articles on herpetology.
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Minh Duc Le has been working on conservation-related issues in Southeast Asia for more than 15 years. His work focuses on biotic surveys, wildlife trade, and the conservation genetics of various wildlife groups in Indochina. Minh is currently working on projects which characterize the genetic diversity of highly threatened reptiles and mammals in the region, and he has pioneered the application of molecular tools in surveying critically endangered species in Vietnam. He has long been involved in studying the impact of the wildlife trade on biodiversity conservation in Vietnam, and is developing a multidisciplinary framework to address this issue in the country.
Thomas Ziegler has been the Curator of the Aquarium/Terrarium Department of the Cologne Zoo, Germany, since 2003. He 1s also the Coordinator of the Biodiversity and Nature Conservation Projects of the Cologne Zoo in Vietnam and Laos. As a Zoo Curator and Project Coordinator, he tries to combine in situ and ex situ approaches, such as linking zoo biological aspects with diversity research and conservation in the Cologne Zoo, in rescue stations and breeding facilities in Vietnam, and in the last remaining forests in Indochina. Since February 2009, he has been an Associate Professor at the Zoological Institute of Cologne University, Germany, and an Adjunct Professor since 2016. Photo by Rolf Schlosser.
Cuong Thien Tran has been working on conservation-related issues in Vietnam for more than 10 years. Cuong 1s currently working on projects which characterize the biodiversity and ecology of Vietnam. In addition, he also carries out work related to environmental education and sustainable development in Vietnam.
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The methodology for rearing the Fire-bellied Toad (Bombina bombina) from protected, small, isolated (but degraded) habitats in its northern distribution range
*’Kristé Stravinskaité, ‘Rasa Jautakiené, ‘Inga Citaviéiené, ‘Alma Pikiniené, ‘Gintaré Stankeviéé 7Vytautas Rakauskas
‘Lithuanian Zoological Gardens, Radvilény rd. 21, LT-50299 Kaunas, LITHUANIA *Laboratory of Fish Ecology, Nature Research Centre, Verkiy str. 98, LT-12201 Vilnius, LITHUANIA *Directorate of Dzikija-Suvalkija Protected Areas, Kampeliy str. 10, Aleknoniy v., LT-64351, Alytus r., LITHUANIA
Abstract.—The Fire-bellied Toad (Bombina bombina Linnaeus, 1761) is a vulnerable and protected species in Europe, where it is suppressed in small, isolated populations in its northern distribution range. The main cause of B. bombina population declines in this region is the loss of suitable habitats due to either anthropogenic factors or natural succession. Recently, very hot summers with prolonged dry and very heated periods have contributed to the declines of B. bombina populations on a very large scale. Therefore, it is important to preserve the natural, although small, populations of B. bombina to save the gene pool of the rare northern populations for the future, which is essential for conservation breeding, research, and outreach with this species. This study provides the rearing methodology, growth rates, and sexual dimorphism of protected B. bombina individuals in their first year.
Keywords. Anura, endangered species, conservation, sexual dimorphism, morphometrics, Natura 2000
Citation: Stravinskaité K, Jautakiené R, Citaviciené |, Pikiniené A, Stankeviéé G, Rakauskas V. 2024. The methodology for rearing the Fire-bellied Toad (Bombina bombina) from protected, small, isolated (but degraded) habitats in its northern distribution range. Amphibian & Reptile Conservation 18(1&2) [General Section]: 47-57 (e332).
Copyright: © Stravinskaité, et al. 2024. This is an open access article distributed under the terms of the Creative Commons Attribution License [Attribution 4.0 International (CC BY 4.0): https://creativecommons.org/licenses/by/4.0/], which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The official and authorized publication credit sources, which will be duly enforced,
are as follows: official journal title Amphibian & Reptile Conservation; official journal website: amphibian-reptile-conservation.org.
Accepted: 20 May 2024; Published: 20 August 2024
Introduction
Interest in species conservation is growing each year, and amphibian species are one of the most endangered groups of vertebrates worldwide (Kurnaz and Kutrup 2019; Stuart et al. 2008; Wake and Vredenburg 2008). The Fire-bellied Toad (Bombina bombina Linnaeus, 1761) 1s one of these vulnerable amphibian species that is distributed mainly in Central and Eastern Europe (Nicoara and Nicoara 2007; Sillero et al. 2014). Bombina bombina is a tailless amphibian. The body of this frog is flat, and its length varies from approximately 40 to 55 mm. The back of B. bombina is dark brown or greyish, and the underbelly is mottled with wide red and orange spots that they use to scare away predators (RimSaité 2021). The most suitable habitats for B. bombina are in lowlands such as shallow fish-free ponds with plenty of sunlight, or shallow edges of larger water bodies (RimSaité 2021; Schroder et al. 2012). This species can also inhabit swamps, flooded
Correspondence. ‘kriste.stravinskaite@zoosodas.It
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ditches, quarries, and peat bogs (Chikhlyaev and Ruchin 2021). Bombina bombina breeds in stagnant, good- quality water with natural eutrophic conditions (Kinne et al. 2006). According to research conducted in Latvia, B. bombina calling males were found in small (<0.5 ha) and medium (0.5—10.0 ha) sized lentic waterbodies and ditches (Ceirans et al. 2020).
The northern range of the distribution of B. bombina in Europe is within northern Lithuania and the south- eastern parts of Latvia (Kuzmin et al. 2008; Pupina and Pupins 2008, 2009). This species is protected in both countries (Berzins 2003; RimSaité 2021). Although the range of B. bombina covers all Lithuanian territory, it has a very fragmented distribution. In the territory of Lithuania, approximately 20,000-—50,000 B. bombina individuals can be found, but there are only a few suppressed populations in the northeastern and western parts of Lithuania. Most of these populations are small, consisting of 10—20 adult individuals (Ivinskis and RimSaite 2011; RimSaité 2021).
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At the beginning of the 20" century, B. bombina was not classified as a rare species in Lithuania (Fedorowicz 1918). Today it is classified as a Near Threatened species, and it is included in the Red Data Book of Lithuania (Rim$Saité 2021). A similar situation is seen in Latvia, where B. bombina populations are very small and isolated (Pupins and Pupina 2012). In most of these populations, no more than 20 vocalizing males were recorded (Pupina 2011; Pupina and Pupins 2007). In Latvia, this species is included in the Red Book of Latvia and assigned to the first endangered species category (Berzins 2003). This is the most recent information described in the literature, but the current situation could be even worse. Furthermore, it is also listed as a protected species in Appendix II of the Bern Convention and Annexes IT and IV of the EU Habitats Directive. Bombina bombina is included in the IUCN Red List of Threatened Species, where its most recent assessment was conducted in 2008 and it was listed as Least Concern (IUCN 2023).
The main causes of declining B. bombina populations in its northern distribution range are the loss of suitable habitats due to either anthropogenic factors or natural succession (Pupina et al. 2018; Pupins and Pupina 2012; Tytar et al. 2018). In some water bodies, populations of B. bombina are reduced by the appearance of the highly invasive, predatory fish species Perccottus glenii Dybowsk1i, 1877 (Pupina and Pupins 2008). In Lithuania, the main drivers of B. bombina decline are changes in land use within the habitats of the species, and climate change leading to prolonged heated periods in the spring and summer (RimSaite 2021). When small ponds dry up, the tadpoles or even adult individuals often die. This can represent a crucial loss for the small, isolated B. bombina populations that are at a very high risk for the loss of genetic variation through genetic drift and inbreeding (Frankham and Ralls 1998). Furthermore, strong shifts in the sex ratios in several isolated populations have been noted, as they have become more isolated during such habitat degradation. Some of these populations became male dominated, while others became female dominated, possibly resulting from the very small numbers of B. bombina individuals in these populations. Therefore, it is important to save these natural, although small, populations of B. bombina in order to stabilize their sex ratio and re-establish them in more suitable habitats for their continuing occurrence within their northern distribution range. To achieve this, it is important to use ex situ methods such as rearing B. bombina individuals in the laboratory and releasing juveniles back into their natural habitats, as they will have a higher chance of survival compared to other stages of development. It is also important to choose suitable ponds for the release of B. bombina individuals, where their survival and breeding in situ in their natural environment would be ensured. Releasing individuals into nature in an equal sex ratio contributes to better survival of the population.
Amphib. Reptile Conserv.
The aim of the study was to develop an effective methodology for the rearing of collected B. bombina spawn from degraded habitats of the small, isolated populations. Several spawn of B. bombina individuals were collected from degraded habitats 1n three locations in Lithuania between 2017 and 2021. Collected spawn was incubated, and the hatched tadpoles were grown and released into more suitable, protected habitats near the initial collection sites. In this article, we provide the rearing methodology, growth rates, and sexual dimorphism of reared B. bombina individuals. This methodology of B. bombina egg-rearing until the juvenile stage may contribute to saving the small, isolated gene pools of B. bombina populations for the future at the northern edge of its species distribution.
Materials and Methods Collection of Bombina bombina Spawn
Spawn of B. bombina was collected from naturally degraded ponds in four Natura 2000 sites in Lithuania: Juodabalé Zoological Reserve (LTLAZ0010); Drapalai village surroundings (LTDRU0004); Kuéiuliske village surroundings (LTLAZO0001); and Margiai village surroundings (LTLAZ0035) (Table 1). Natura 2000 is an ecological network of rare and threatened species protection sites, which stretches across all 27 European Union countries. Spawn was collected during the end of spring and beginning of summer (May—June), when water temperatures reached 13-14 °C, in 2017, 2018, 2020, and 2021. The year 2019 was not included in the study, because that spring was very hot and dry which caused the total drying out of the B. bombina spawn. The spawn of eggs was carefully collected from flooded plants at the earliest stage of their development and placed in a 10 L bucket half-filled with pond water. Water from the ponds was taken along with the resident microfauna so that the tadpoles could feed in the early stages. The buckets with eggs were kept outdoors to protect them from strong temperature fluctuations, and at 1-2 days after collection, they were delivered to the Lithuanian Zoological Gardens (Kaunas, Lithuania).
Incubation of Spawn
The delivered spawn with water was very carefully transferred to 60 L plastic containers (Fig. 1A) that were filled with water to three-fourths capacity. The water pH was 7.6—7.8, and total water hardness was 2.6—2.8 mmol/L. In the laboratory, the initial water temperature was maintained similar to outdoors (13—14 °C), and within 5 days the temperature was gradually increased to 24—25 °C and maintained at that level for the remainder of the study. The temperature was increased gradually to avoid thermal shock to the eggs. Overall, the average water temperature was maintained
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Fig. 1. Bombina bombina rearing system. (A) Plastic rearing tanks. (B) Three-month-old B. bombina individuals. (C) Eight-month-old B. bombina individuals.
at 21.0 + 2.3 °C during all incubation periods. Before filling the containers, they were washed with antiseptic and rinsed thoroughly with hot water. The lids of containers were with perforated with holes. The larvae hatched in 5-7 days.
Rearing during the Larval Period
After hatching, the B. bombina tadpoles were kept in 60 L plastic containers with dimensions of 58 x 35 x 30 cm. Every day, dirt was removed from the bottom with a sieve, and one-fifth of the water was drained, and fresh tap water was added slowly. The water with larvae was moved as little as possible. The water pH was 7.6—7.8, and total water hardness was 2.6—2.8 mmol/L. No water aeration or filtration was used. The average water temperature in the containers was maintained at 23.8 + 1.1 °C during all metamorphosis stages. We evaluated the embryonic development according to the stages described by Gosner (1960), and the 23-25 stage of B. bombina development was reached in 3—5 days. At weekly intervals up to 3 months of age, the juveniles were grouped by their stage of development because their rate of development varied. Before metamorphosis was complete, when the tadpoles entered the 4-leg stage, they needed a place to climb, rest, and put their head above the water (.e., land). They were transferred to 35 L plastic tanks with a water level of about 5—7 cm and parts of land without water. The tanks were placed at an angle of about 35—
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45°. Dried oak leaves, live plants, stones, and other materials were placed in the water to make it easier for the juveniles to reach the land. The tanks were covered with dense gauze of 2 x 2 mm mesh size held in place with a rubber band because the juveniles could escape. Lighting was provided by 28W incandescent lamps and REPTI PLANET Repti UVB 2.0 13W (Czech Republic) lamps installed 20 cm above the tanks. The UVB-containing lamps were turned on every day for about 4 hours. UVB rays stimulate the production of vitamin D and calcium uptake (Michaels et al. 2015). The length of the photoperiod was similar to the natural rhythm of day and night, and it was adjusted according to the season.
During metamorphosis, it was important to keep enough microfauna in the water for the tadpoles to feed upon. Microfauna was collected along with the water during egg collection. Tree leaves that began to decay were added to the water so that microorganisms (such as Paramecium genus) could begin to proliferate. Dry oak leaves were added to the water due to their disinfectant properties and soft leaves of oak, willow, apple, and pear trees, along with blanched nettles, provided more food for the tadpoles. At the beginning, they were also fed daily with dry feed for fish fry Vipan Baby (Sera, Germany), and later with Bio-vit (Tropical, Poland).
The full larval development period of B. bombina lasted for about 45-65 days. Metamorphosis was completed when the juveniles completely lost their tail and had four fully grown legs.
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Table 1. The locations (as the codes for LT Habitat Protection Important Territories) of B. bombina spawn collection and where reared individuals were released during the study period (2017-2022).
Collection date Locations of collected spawns
2017 06 29 LTDRUO004 2018 05 04
LTLAZ0010 2018 05 15 2020 05 30
LTLAZ0010 2020 06 02
LTLAZ0010 2021 05 13 LTLAZ0001
LTDRUO004
Post-metamorphosis Rearing
After they had completed metamorphosis, the 3-month- old juveniles were transferred to 60 L plastic tanks (Fig. 1B). The juveniles were grouped into three tanks according to size, in order to reduce interspecific competition, with 10 to 15 juveniles in each tank. The smallest juveniles were transferred to 10 L tanks, with a lot of vegetation (Epipremnum spp., Anubias spp., and Cryptocoryne spp.) to allow the juveniles to get out of the water. In these tanks, the water depth was about 2-4 cm. All the water in the tanks was changed at least twice a week, or more often if necessary. The average water temperature in the containers was maintained at 23.8 + 0.6 °C during all post-metamorphosis periods. During post-metamorphosis rearing in the tanks, UVB-containing lamps were turned on every day for about 4 hours.
The juveniles were fed daily with an amount of food that corresponded to about 10-12% of their body weight. The first food source given to juveniles was live fruit flies (Drosophilidae), which were added to the rearing tank. House Crickets (1- to 3-day-old Acheta domesticus) and Two Spotted Crickets (1- to 5-day-old Gryv/lus bimaculatus) were given to very small and weak individuals. From 5 to 7 flies were provided for each juvenile, and the uneaten fruit flies were fed on carrot slices, cabbage leaves, etc. Vitamins and minerals were dusted on the fruit flies twice a week. Small crickets (Gryllus sp.) were added to the diet of one-month-old juveniles. Live mosquito larvae (Chironomus sp.) were added to the diet of juveniles at 6—7 weeks old. From the age of 2 months, juveniles were fed Turkestan Cockroaches (Shelfordella tartara). All feed was provided live.
Wintering Period of Rearing Juveniles The overwintering period was carried out in the lab because the seasonal temperature variation is important
for the development and phenology of temperate
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Release date Locations of release
2018 06 22 LTLAZ0010
2019 06 14 LTLAZ0010
LTLAZ0010
2021 06 11 LTLAZO0001
LTDRUO004
2022 06 17 LTLAZ0035 amphibians, especially their reproductive cycles.
Five-month-old B. bombina juveniles were prepared for wintering. Over a period of two weeks, the water temperature was gradually reduced to 10 °C and the photoperiod was reduced to 5 hours. The amount and caloric content of the food was also reduced gradually by 20% per day for five days in a row, and starting at two days before the wintering period, juveniles were no longer fed at all. This was done to ensure that the guts of overwintering toads were empty, otherwise the food inside would rot. Plastic tanks (15 L) with moist 5.5-—6.5 pH neutralized peat (DURPETA, Lithuania) and softened dried oak leaves were prepared for the wintering of B. bombina juveniles. This substrate was placed in tanks at a depth of 5—7 cm. Ten to 15 juveniles were placed in the tanks for wintering. The wintering tanks were transferred to a wintering laboratory where the water temperature was maintained at 6-8 °C and checked daily. The wintering laboratory was locked, and the results of the temperature sensors were observed without entering the laboratory, in order to avoid causing any additional sounds. The laboratory was lit by a faint blue light. The condition of the juveniles was checked once a month and the B. bombina juveniles wintered for 12 weeks.
Post-wintering Period of Rearing Juveniles
At the end of wintering, the juveniles (now six months old) were transferred to the laboratory, where the temperature was 10 °C. After wintering, when the temperature reached at least 15 °C, the B. bombina juveniles were fed minimally with fruit flies. Later, the amount and caloric content of the food was gradually increased. Over one week, the water temperature was gradually raised to 17 °C. The photoperiod was extended to 8 hours. The juveniles were kept in plastic tanks for two more months until they reached 8 months of age (Fig. 1C), and then 20 individuals were placed
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into each 100-liter aquarium with a 2—3 cm thick layer of gravel (2-5 mm diameter). The water level in the aquariums was about 10 cm. On the bottom of the aquariums, the substrate consisted of stones and other items, and artificial or live plants were added. The aquariums were equipped with automatic thermostats and filters with air compressors. Above the tanks were lighting fixtures with 28 W incandescent lamps and UVB 2.0 lamps. After full recovery, the average water temperature in the containers was maintained at 23.7 + 0.5 °C during all post-wintering periods. Each time the water was changed, its level was raised by 1-2 cm to a final depth of 17—22 cm.
After recovery from wintering, all juveniles were divided into three equal groups according to weight to avoid interspecific competition. The separate groups were not mixed until their sex was determined, and they were released back into the wild.
Juvenile Rearing in Outdoor Tanks
When the maximum outdoor air temperature reached approximately 21—23 °C during the day, B. bombina individuals were moved outside. They were placed into 1.5 m? tanks covered with a fine net. In rainy weather, the tanks were covered with lids that had air vents. The bottoms of the tanks were covered with pebbles, and water plants were planted under the water. On cold nights (when the outside temperature dropped below 15-18 °C), the thermostats were switched on. In general, animals that will be reintroduced into nature must have as little contact with humans as possible. For this reason, the B. bombina individuals were not exhibited to visitors of the Lithuanian Zoological Gardens where they were reared.
Environmental Conditions and Promotion of Natural Behavior during Post-metamorphosis
After metamorphosis, the B. bombina juveniles lived in a more sterile environment than they would have experienced in nature. However, the soil, stones, oak leaves, live aquatic plants, and other natural materials provided hiding places and created an environment similar to nature. The juveniles would try to hide when they felt threatened.
Another even stronger way to promote the natural behaviour of the juveniles was to let them search for live food, 1.e., to hunt for it. As mentioned above, the diet included live fruit flies, mosquito larvae, various cockroaches, and crickets. Competitive relations emerged between the B. bombina juveniles. In adaptation tanks, the juveniles were placed in a small area similar to their natural environment. There B. bombina juveniles could swim freely, and climb over the branches, rocks, or plants which were above the water.
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Sex Determination and Morphometric Measurements
The sex of B. bombina individuals was determined as they matured (at 9 months old, after the full metamorphosis). In addition to sex identification by nuptial pads, croaking males were noted to confirm their sex. Moreover, some of the females spawned before their sex had been determined.
Total body weight and the two morphometric parameters of body (snout-vent) length (SVL) and sacral width (SW) were measured for the reared B. bombina individuals before they were released into the wild. Morphometric measurements were taken using a digital calliper (Carbon Fiber Composites, model CTCF1506, China; resolution 0.1 mm, accuracy + 0.1mm). The 1 mm margin of error in length and width measurements was permissible in order to avoid traumatizing the animals. The weight of juveniles was measured using scales (Romansas, model KB, Lithuania; accuracy 0.01 g and error + 0.1 g).
Transporting the Animals and their Release into Nature
At9 months of age, depending on the weather conditions, the B. bombina juveniles were handed over to the workers responsible for the protected areas for release into the wild. Juveniles were not fed for one day prior to being released into the wild. In the evening before release, juveniles were brought from the adaptation tanks and kept 2 degrees cooler. It was very difficult to predict the outdoor air temperature on the following day, and if the juveniles are abundantly fed and the air temperature cools suddenly, the feed can begin to spoil and kill the juveniles. In the morning, 10-15 B. bombina juveniles were placed in pre-made 15 L plastic boxes. The bottoms of the plastic boxes were covered with a 2 cm thick layer of moss (Sphagnum sp.) and a small amount of water to minimize the stress of the juveniles during transportation. Juveniles were released in pre-arranged Habitat Protection Important Territories (HPIT) of the Natura 2000 network territories in Lithuania (Table 1). The number of B. bombina juveniles to be released into each water body was arranged with the responsible personnel of the protected areas before the date of release.
Statistical Analyses
For collating the data, all measured juveniles were divided into two groups according to their gender, which was derived from their sexual behavior. The gender effects on the weight and measurements of snout-vent length (SVL) and sacral width (SW) of juveniles were estimated by two-way ANOVA, also including a year factor and their interaction. In all three cases, the
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model residuals did not significantly deviate from a normal distribution (Shapiro-Wilk’s tests: p > 0.05). The analyses were performed using STATISTICA 12.0 software. The significance level of p < 0.05 was specified for all statistical analyses a priori.
Results Survival
In total, 229 tadpoles developed from the collected spawn during the study period. The proportion surviving to successful metamorphosis and the overall percentage of specimens that reached the adult stages were very high at 93.2 and 92.4%, respectively (Table 2). However, there were some considerable differences in the metamorphosis success of tadpoles between the years. The first two years showed the lowest survival rate, with very low numbers in 2018. That year tadpoles were reared in tanks with water filters, which made an effect of running water, while in later years the hatched tadpoles were grown in still water. After methodical changes in tadpole rearing, the success rate increased remarkably (Table 2).
Growth and Sexual Dimorphism
In nine months, the reared B. bombina individuals reached 38.9 + 2.5 mm in body length, 22.7 + 3.3 mm in body width, and they weighed 7.4 + 1.3 g. The total body weight, length, and width indicating the growth of juveniles varied between males and females in the different years (Table 3). The available data allow for statistical testing of a simple hypothesis. Significant differences were observed in all three measurements between males and females, and females were bigger (Table 4). However, only the SVL and SW measurements varied significantly between the different years, while the comparison of different study years did not reveal any difference in juvenile weight (Table 4).
A retrospective comparison of the reared B. bombina juveniles revealed gender differences in their weight. Each year, the reared juveniles were grouped into three equal groups according to their total body weight after six months (Table 5). The sexing of the adults showed that the first juvenile weight group included almost 95% of the females. While the third group included almost entirely (90%) males. Gender was distributed almost equally in the middle group, at 43% male and 57% female. Overall, the results showed that the gender of six-month-old juveniles could be ascertained by separating them into three groups according to size even before they had truly matured. In this case, the gender could be assigned with more than 90% accuracy within the first (largest) and last (smallest) groups of separated juveniles (Table 5).
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Discussion
Bombina bombina is considered to be of Least Concern globally (UCN 2023), but it is also important to prevent local extinction. This species is distinguished as the targeted protected species in 31 Habitat Protection Important Territory (HPIT) areas of the Natura 2000 network territories in Lithuania (SRIS 2023). However, the significant decline of small, highly fragmented B. bombina populations was recently observed within the territory of Meteliai HPIT of the Natura 2000 network. In recent years, B. bombina individuals, and especially their spawn, have been dying due to prolonged late spring or summer droughts, during which the spawn laid in their usual reproduction habitats have dried up. Bombina bombina females usually spawn in shallow ponds on the stems of vertical water plants (RimSaiteé 2021). Each year, natural ponds are full of water at the end of spring, but then the water level drops suddenly, and the eggs die before they can hatch; and even if some of the tadpoles do hatch, they die as the water level drops.
Furthermore, fragmented B. bombina populations are declining because of accelerated natural succession, after remarkable changes in surrounding land use have occurred. In recent years, small water ponds that were once suitable for B. bombina breeding have become overgrown with grass, ponds are becoming shady, or they are being deepened for the development of extensive fisheries which cause dramatic habitat changes and fragmentation (RimSaite 2021). Moreover, the rapidly decreasing number of small farms at the country scale is leading to the destruction of many small ponds, which had been used as the drinking water source for farm animals and were also very suitable habitats for B. bombina individuals to thrive (RimSaité 2021). As a result, many B. bombina habitats with previously known populations are disappearing. Therefore, to protect the remaining small and protected B. bombina populations, our aim was to create a methodology for the collection and incubation of spawn, and post-metamorphosis tadpole rearing based on the environmental conditions where their development is highly threatened.
Spawn of B. bombina was collected and _ the subsequently reared individuals were released in the four HPITs (Table 1). Bombina bombina spawn was collected from specific water ponds where they had no chance for survival, while the reared specimens were released to either a different HPIT or the same HPIT, but in different watercourses with a good state for maintaining viable B. bombina populations. These watercourses are deep enough for B. bombina to successfully survive and reproduce, they do not dry up during hot spring or summer months, and do not become overgrown with grass. The release of B. bombina individuals in suitable water ponds will increase natural local populations of the species in the Natura 2000 protected sites. By increasing the B. bombina populations in these areas, the species has
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Table 2. Numbers of developed B. bombina tadpoles, individuals after metamorphosis, and individuals released to water ponds from 2017-2022.
Egg Number of Number of Percentage of Numbe of Feueniaee ee Pie re el individuals of overall collection developed individuals surviving individuals surviving Release year ' released to survival of year tadpoles after metamorphosis after metamorphosis Pines ponds individuals 2017 69 64 92.8% 2018 61 88.4 % 2018 56 31 55.4 % 2019 31 55.4% 2020 70 70 100.0 % 2021 70 100.0 % 2021 73 67 91.8% 2022 67 91.8% Total 268 232 93.2% 229 92.4%
Table 3. Numbers of male and female B. bombina juveniles and their mean weight values, lengths and widths from 2018-2022. Note: n— number of individuals; F — female; M — male; SD — Standard deviation
Year n Weight Length Width F M F M F M F M Mean +SD Mean SD Mean +SD
2018 20 36. 79407 6.3+0.8 38.741.9 36.8 42.2 2462 220 19.7+1.0
2019 14 17. 7.7408 6.2+0.8 59 2227 36.94 1.9 24.9+2.0 194409
2021 39 31 8.2+0.9 6.7+0.5 40.6+2.2 38.342.1 26.142.4 20.44 1.1
2022 44 23 8.0+41.7 6.6+0.4 40.2 + 2.0 38.74 1.7 25.04 2.3 19.8+1.8 Mean 122 107. 8.0+0.9 6.8 + 1.6 39.9422 37.8465 25.223 21.9448
Table 4. Results of two-way ANOVA testing for the effects of gender (male vs. female) and study year on B. bombina weight, length, and width measurements.
Measurement Factor df F 72) Length (SVL) Gender l 45.83 < 0.001 Year 3 10.62 < 0.001 Gender = Year 3 0.45 0.72 Error 221 Width (SW) Gender 1 403.47 < 0.001 Year 3 6.08 < 0.001 Gender x Year 3 1.25 0.29 Error 221 Weight Gender 1 187.02 < 0.001 Year 3 2.61 0.05 Gender = Year 3 0.83 0.48 221
Error
Table 5. Separated weight groups of six-month-old B. bombina juveniles and their gender distribution within the resulting groups.
Grou "i Weigh Male Female P mean + SD number (%) number (%) I LS 3.8403 4 (5.3 %) 71 (94.7 %) II 75 3 lt 0:2 32 (42.7 %) 43 (57.3 %) Ill 79 25-2033 71 (89.9 %) 8 (10.1 %) Amphib. Reptile Conserv. 53 August 2024 | Volume 18 | Number 1&2 | e332
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its best chance for survival, and this is a way to save this locally endangered species from extinction.
During this study, we considered biosecurity and took all necessary precautions to reduce the risk of accidentally introducing pathogens along with the released B. bombina. A separate laboratory was built for B. bombina rearing to eliminate the risk of accidental contamination or transport of pathogens from other laboratories. Lab coats were required for working in the laboratory, and only people who worked on this project were allowed to enter the laboratory. Everyone who worked in the laboratory was familiar with the laboratory and biosafety protocols. Separate gloves, aquariums and cleaning equipment were used for each tank in order to avoid any possible contamination of pathogens from other tanks. For tank and laboratory disinfection, a saturated solution of table salt was used because B. bombina individuals are very sensitive to disinfectants and cleaning agents. Also, individuals were removed from the tanks during cleaning and disinfection. After disinfection, the tanks and laboratory were carefully washed with water in order to avoid leaving any residues of the disinfectant. Water in the tanks of B. bombina juveniles was changed daily because it gets contaminated quickly with leftover food and animal excreta. Tanks were filled only with fresh clean room temperature water.
In total, 229 tadpoles developed from the collected spawn during the study period. The percentage of B. bombina individuals which survived after metamorphosis (93.2%) and the overall survival of individuals (92.4%) during this study were very high (Table 2). The observed survival rate of tadpoles in natural habitats is only 6%. The survival percentage in this study was higher than 88% in all years except 2018. That year the tadpoles were grown under different conditions. The tadpoles were transferred to 120-liter aquariums in which the water level depth was about 25—30 cm. The aquariums were filled with live aquatic plants and 5—7 mm sized river pebbles, and water filters produced an effect of running water. The tadpoles began to die a month after they were transferred to these aquariums. The tadpoles were transferred from these unsuitable conditions to 60-liter plastic containers with live aquatic plants and river pebbles, in which water was standing still and its level was maintained at about 30 cm. After the growing conditions were changed the tadpoles stopped dying. During the subsequent years, the survival percentage of the tadpoles increased from 91.8% to 100% (Table 2). These results show that our egg incubation, rearing, and feeding methodology is effective and can be used for rearing this species in artificial conditions and releasing the adults into their natural habitats.
The survival results of B. bombina breeding under the laboratory conditions reported by other authors are consistent with our findings. Earlier studies showed mortality rates of tadpoles from 4 to 7% and juveniles at 8% (Kinne et al. 2006). In this study, the overall tadpole mortality was 6%, while for juveniles, mortality was
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only 1%. As expected, the survival rate under artificial conditions was much higher than natural conditions because the factors important for the successful development and survival of B. bombina were kept stable, such as air and water temperature, water level, and the amount of food. Moreover, the individuals experienced only intraspecific competition for food or habitat, and they did not experience interspecific competition since they were grown without any other species present. There were no predators or species with which they needed to compete for food. In natural habitats, such interspecific competition is unavoidable, and the living conditions are not very stable. Furthermore, the artificially reared B. bombina individuals were repeatedly grouped by their size and kept in rather small numbers to reduce the intraspecific competition for food and preferred habitat.
Innature, B. bombina individuals reach sexual maturity at 2-4 years (Bulbul et al. 2018). Mature individuals can reach a snout-vent length of 56 mm (Lang 1988), but usually it ranges from 40 to 55 mm (Rim$aité 2021). The B. bombina individuals reached maturity much earlier in the artificial conditions of this study than in their natural habitats. Our findings revealed that B. bombina individuals reached maturity at 8-9 months, when the males began vocalizing and some females spawned. The mean snout-vent lengths were 39.9 + 2.2 mm for females and 37.8 + 6.5 mm for males (Table 3). By comparison, the observed mean SVL of adult female B. bombina individuals was lower in some Romanian populations, e.g., 35.2 mm (Cogalniceanu and Miaud 2004) and 36.8 mm (Cogalniceanu and Miaud 2003). However, it was higher (47.1 mm) in Polish populations (Rafinska 1991). The mean SVL of male B. bombina individuals from the Romanian populations was also lower compared to our results, at 34.4 mm (Cogalniceanu and Miaud 2004) and 36.6 mm (Cogalniceanu and Miaud 2003).
Sexual size dimorphism (SSD) is present in over 90% of amphibian species (Nali et al. 2014), although it is weakly expressed in B. bombina (Cogalniceanu and Miaud 2004; Bulbul et al. 2018). Males have nuptial pads, which appear only in sexually mature individuals before the breeding season (Bulbul et al. 2018). However, we found a significant difference in measured SVL and SW between B. bombina males and females (Table 3). The weight of B. bombina juveniles also showed significant differences between the sexes. Female individuals were heavier than males. Incontrastto our results, Cogalniceanu and Miaud (2004) in Romania and Bulbul et al. (2018) in Turkey found that adult B. bombina individuals in natural habitats did not show any significant differences in body mass or SVL between the sexes. A significant correlation was found between SVL and the age of a B. bombina population in Romania (Cogalniceanu and Miaud 2003).
The sex of amphibians is determined genetically, and many studies have shown that high temperatures result in male-biased populations, while female-biased populations develop in low temperatures (Cogalniceanu
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and Miaud 2003). The number of males per pond is usually less than 10 in the northern distribution countries such as Latvia and Lithuania (Kuzmin et al. 2008). An unbalanced sex ratio affects the size of the population. Our rearing methods allowed us to predict the sex of six- month-old juveniles and individuals could be distributed into groups by their sex before they were mature. The multiyear threshold of B. bombina juvenile weight may be used for sex determination with >90% accuracy. This is very useful because this method enables the release of individuals into natural ponds with an equal sex ratio even before they are mature. Such knowledge provides a valuable tool for the management of a declining population by balancing the unbalanced sex ratio in naturally threatened B. bombina populations.
Overall, the conservation methods presented here can allow us to save small vulnerable populations of B. bombinaand prevent them from extinction by maintaining their genetic diversity and releasing individuals into more favorable habitats that are usually unavailable due to their very segregated distribution. Such efforts will help preserve the declining populations of the species at the northern edge of their range for future generations and prevent this species from complete extinction where their habitats are much too fragmented.
Acknowledgments
This study was funded by the project “Implementation of Nature Conservation and Management Measures by Preserving and Increasing the Populations of European Pond Turtles and European Fire-bellied Toad” (Nr. 05.4.1-APVA-V-018-01-0004).
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Pupina A, Pupins M. 2009. Comparative analysis of biotopes and reproductive-ecological manifestations of Bombina bombina (Linnaeus, 1761) in Latvia. Acta Biologica Universitatis Daugavpiliensis 9(1): 121-130.
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Pupina A. 2011. Sarkanvédera ugunskrupju Bombina bombina L. ekologijas Tpatnibas uz sugas areala ziemelu robezas_ Latvija. Ph.D. Dissertation, Daugavpils Universitate, Daugavpils, Latvia.
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RimSaite J. 2021. Radonpilvé kimute. Bombina bombina (Linnaeus, 1758). Pp. 207 In: Red Data Book of Lithuania. Animals, Plants, Fungi. Editor, RaSomavicius V. Lututé, Vilnius, Latvia. 684 p.
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SRIS. 2023. Saugomy rtSiu informaciné sistema (Information system of protected species). Available: https://sris.am.It/ [Accessed: 2 May 2023].
Stuart S, Hoffmann M, Chanson J, Cox N, Berridge R, Ramani P, Young B, Editors. 2008. Threatened Amphi- bians of the World. Lynx Edicions, Barcelona, Spain; IUCN, Gland, Switzerland; and Conservation International, Arlington, Virginia, USA. 134 p.
Tytar V, Nekrasova O, Pupina A, Pupins M, Oskyrko O. 2018. Long-term bioclimatic modeling of the distribution of the Fire-bellied Toad, Bombina bombina (Anura, Bombinatoridae), under the influence of global climate change. Vestnik Zoologii 52(4): 341-348.
Wake DB, Vredenburg VT. 2008. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences of the United States of America 105: 11,466-11,473.
Wojdan D. 2010. Impact of vehicle traffic on amphibian migrations in the protection zone of the Swietokrzyski National Park. Teka Komisji Ochrony i Ksztaltowania Srodowiska Przyrodniczego 7: 466— 472.
Kristé Stravinskaité is a Scientific Curator at the Lithuanian Zoological Gardens and a Ph.D. student at Vytautas Magnus University in Kaunas, Lithuania. She obtained her M.Sc. degree in Molecular Biology from Vytautas Magnus University in Kaunas, Lithuania. Kristé has been working in the scientific field for 11 years, mainly conducting genetic research on several invasive species. Her research interests are diverse, but focused on the conservation of biodiversity, endangered species, genetics, and ecology. Kristé regularly attends biodiversity conferences and is the co-author of several scientific
Rasa Jautakiené is an Ectotherm Care Specialist at the Lithuanian Zoological Gardens and obtained the qualification of a veterinarian at the Lithuanian Veterinary Academy. Rasa breeds various rare species of amphibians. Since 2010, she has been actively working as a specialist on conservation projects involving Lithuanian reptiles and amphibians. Working as a Project Specialist, she was responsible for creating the right conditions and monitoring the growth of Bombina bombina in the laboratory. Rasa is the co-author of the Bombina bombina breeding methodology.
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Vytautas Rakauskas is a Senior Researcher at the Nature Research Centre in Vilnius, Lithuania. He obtained his Ph.D. in 2014 from the Nature Research Centre and Vilnius University, Lithuania. From 2010 to 2018, he was teaching three courses of “Ecology of Hydro-ecosystems,” “Hydrobiology,” and “Zoology of Vertebrates” at Vilnius Uni- versity. His research focuses on the non-indigenous species and freshwater ecosystems in Europe. Vytautas is the co-author of one book and more than 25 papers related to ecology and biodiversity in Europe.
Inga Citavitiené is a Chief Specialist at the Biodiversity Protection Department of the Directorate of Dztkija-Suvalkija Protected Areas, Alytus, Lithuania. She obtained a B.Sc. degree in Ecology and Environmental Science from the Lithuanian University of Agriculture. Inga has been working in the protection and management of biodiversity for 18 years and conducts biodiversity monitoring.
Alma Pikiniené is an Ectotherm Curator at the Lithuanian Zoological Gardens, has a M.Sc. degree in Biology from Vilnius University, and has more than 30 years of experience working at the zoo. Since 2010, Alma has been actively working as a Specialist/ Coordinator for conservation projects with Lithuanian reptiles and amphibians. Working as a Curator and Project coordinator, she combines in situ and ex situ methods, thereby contributing to the restoration of populations of Bombina bombina, Emys orbicularis, and other species. Alma is a co-author of several scientific articles on herpetofauna breeding methods.
Gintaré Stankeviéé is a Director at the Lithuanian Zoological Gardens and Ph.D. student at the Lithuanian University of Health Sciences, Faculty of Animal Science in Kaunas, Lithuania. She has been working in the scientific field for eight years, focusing on livestock, domestic, and wild animal breeding and nutrition. Her research interests are diverse, but most recently Gintaré has been concentrating on the conservation of biodiversity and endangered species.
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Amphibian & Reptile Conservation 18(1&2): 58-67 (e333).
Official journal website: amphibian-reptile-conservation.org
urn:Isid:zoobank.org: pub:3898BB8C-894A-4C1 B-B7C4-D9814E6E8057
A new species of Sipo Snake, Chironius (Serpentes: Colubridae), from the Yungas of Bolivia
"Oliver Quinteros-Mufioz, ‘Pedro Gomez-Murillo, '?Teresa Camacho-Badani, *Rodrigo Aguayo, 3Rene Carpio-Real,‘Edson Pérez, *Bladimir Marca, *°Lucindo Gonzales, and 2*Omar Torres-Carvajal
'Museo de Historia Natural Alcide d’Orbigny, Casilla 843, Cochabamba, BOLIVIA *Museo de Zoologia, Escuela de Ciencias Bioldgicas, Pontificia Universidad Catélica del Ecuador, Avinida 12 de Octubre y Roca, Apartado 17-01-2184, Quito, ECUADOR ?Centro de Bioversidad y Genética, Universidad Mayor de San Simon, Casilla 538, Cochabamba, BOLIVIA *Independent Researcher. C/Rafael Canedo 310, Cochabamba, BOLIVIA *Parque Nacional Carrasco, Av. Santa Cruz N. 1737, Cochabamba, BOLIVIA °Museo de Historia Natual Noel Kempff Mercado, Avenida Irala 565, Santa Cruz, BOLIVIA
Abstract.—A new snake of the genus Chironius is described based on external morphological characters and phylogenetic evidence. The new species occurs in Bolivia, both in the humid montane forests of the Yungas of Cochabamba and in Santa Cruz. It differs from all congeners in having 10 dorsal scale rows at midbody, an entire cloacal plate, keeled paravertebral rows, lightly colored lower portions of the supralabials, a yellow snout, a short hemipenis, and lacking postocular stripes, proximal enlarged spines on the hemipenis, and apical pits. Adults and juveniles have an emerald green background color. The new species is recovered as the sister taxon of C. Jeucometapus, which is known from the Amazonian slopes of the Andes between central Peru and northern Ecuador. We also provide an identification key to the species of Chironius with 10 dorsal
rows at midbody.
Keywords. Carrasco National Park, hemipenes, phylogeny, reptiles, Squamata, systematics
Citation: Quinteros-Mufhoz O, Gédmez-Murillo P, Camacho-Badani T, Aguayo R, Carpio-Real R, Pérez E, Marca B, Gonzales L, and Torres-Carvajal O. 2024. A new species of Sipo Snake, Chironius (Serpentes: Colubridae), from the Yungas of Bolivia. Amphibian & Reptile Conservation 18(1&2): 58-67 (e333).
Copyright: © Quinteros-Mufioz et al. 2024. This is an open access article distributed under the terms of the Creative Commons Attribution License [Attribution 4.0 International (CC BY 4.0): https://creativecommons.org/licenses/by/4.0/], which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The official and authorized publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation; official journal website: amphibian-reptile-conservation.org.
Accepted: 3 July 2024; Published: 2 October 2024
taxonomic work, nine species of Chironius are currently recognized in Bolivia (Embert 2007; Wallach et al. 2014): C. exoletus Linnaeus, 1758, C. flavolineatus Jan,
Introduction
The genus Chironius Fitzinger, 1826 currently contains
24 terrestrial or semi-arboreal species, five of which have been described in the last 15 years (Entiauspe- Neto et al. 2020; Fernandes and Hamdan 2014; Hamdan and Fernandes 2015; Kok 2010; Sudré et al. 2024). Commonly known as Sipo Snakes, they are unique among Neotropical snakes in having 10 or 12 longitudinal rows of dorsal scales around the midbody (Dixon et al. 1993; Hollis 2006). Species of Chironius occur from Honduras in Central America to Uruguay and Argentina in South America (Dixon et al. 1993). The monophyly of Chironius is strongly supported by both morphological and DNA-sequence data (Hamdan et al. 2017; Hollis 2006; Klaczko et al. 2014; Torres-Carvajal et al. 2019b).
Following the comprehensive taxonomic revision of Chironius by Dixon et al. (1993), along with subsequent
1863, C. fuscus Linnaeus, 1758, C. laurenti Dixon, Wiest, and Cei, 1993, C. maculoventris Dixon, Wiest, and Cel, 1993, C. monticola Roze, 1952, C. multiventris Schmidt and Walker, 1943, C. guadricarinatus Boie, 1827, and C. scurrula Wagler, 1824. Recent herpetological field work in the Bolivian-Peruvian Yungas of Carrasco National Park (Cochabamba, Bolivia) and the Tucuman-Bolivian Yungas of Santa Cruz yielded several specimens of Chironius that could not be assigned to any of the described species. Herein we describe them as a new species based on morphological and _ phylogenetic evidence.
Materials and Methods
Specimen Sampling and Morphological Data
Correspondence. ‘ohlisin@gmail.com (OQM); lotorres@puce.edu.ec (OTC)
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In addition to the type series listed below, specimens examined in this study are listed in Appendix 1. Specimens were collected under permits MMAyA- VMA-DGBAP-004 and VMABCCGDE N° 16/19 issued by Ministerio de Medio Ambiente y Aguas and sacrificed in the field by intracardiac injection of 1% xylocaine solution. Immediately after euthanasia, muscle tissue samples were extracted from all specimens and preserved in 96% ethanol. Specimens were subsequently fixed in 10% formalin, stored in 75% ethanol, and deposited in the Bolivian herpetological collections of the Museo de Historia Natural Alcide d’Orbigny (MHNC-R), Cochabamba, and Museo de Historia Natural Noel Kempff Mercado (MNKR), Universidad Autonoma Gabriel René Moreno, Santa Cruz de La Sierra.
We followed the terminology proposed by Dowling (1951) and Dixon et al. (1993) for scale counts and measurements. Snout-vent length and tail length were recorded using a measuring tape to the nearest 0.1 mm. All other measurements were made with digital calipers to the nearest 0.01 mm (rounded to the nearest 0.1 mm) and included: head width (HW), at widest point of head between mouth commissures; head length (HL), from tip of rostral to posterior border of last supralabial;: horizontal eye diameter (EL); eye-nostril distance (EN), from posterior border of nostril to anterior corner of eye; snout length (SL), from rostral tip to anterior margin of eye; maximum loreal length; and maximum loreal height. The morphological and hemipenial terminology follows Dowling and Savage (1960) and Dixon etal. (1993). Color was recorded for both living and preserved specimens. Maxillary teeth were counted in situ on the right side. Sex was determined by searching for hemipenes either by eversion or through a ventral incision at the base of the tail. We prepared the left hemipenis of paratype MHNC-R 3229 (SVL = 810 mm) following standard techniques (Pesantes 1994). We compared data from the specimens examined with literature data from Dixon et al. (1993) and Torres-Carvayal et al. (2019a).
DNA Data and Phylogenetic Analyses
We obtained new DNA sequences from the holotype of the new species described here for three mitochondrial genes, the small (/2S, 424 aligned bp) and large (/6S, 444 aligned bp) ribosomal subunit genes, and subunit [V of NADH dehydrogenase (ND4, 693 aligned bp), as well as one nuclear gene, the oocyte maturation factor mos (c-mos, 537 aligned bp). The muscle tissue sample was mixed with Proteinase K and lysis buffer and digested overnight. Total genomic DNA was extracted using a guanidinium isothiocyanate extraction protocol. DNA samples were quantified using a Nanodrop® ND-1000 (NanoDrop Technologies, Inc.), re-suspended, and diluted to 25 ng/ul in ddH,O prior to amplification. Primers and amplification protocols follow Torres- Carvajal et al. (2019b), and we added the new sequences
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to the dataset in that study to produce a matrix of 173 taxa and 2,098 aligned nucleotides. GenBank accession numbers of the sequences produced in this study are PP408265 (/2S), PP408266 (/6S), PP411803 (ND4), and PP411804 (c-mos).
Data were assembled and aligned in Geneious Prime 2022.1.1 (https://www.geneious.com) under default settings for MUSCLE 3.8.425 (Edgar 2004). Protein- coding sequences were translated into amino acids for confirming the alignment and absence of pseudogenes. After partitioning the concatenated dataset by gene, we ran a maximum likelihood analysis in RAXML v8.2.10 (Stamatakis 2014) under the GTRGAMMA model for each partition. We assessed nodal support with the rapid bootstrapping (BS) algorithm (Stamatakis et al. 2008) on 1,000 replicates. We executed these analyses in the CIPRES Science Gateway (Miller et al. 2010). Outgroup taxa were the same as in Torres-Carvajal et al. (2019b). We used DIVEIN (Deng et al. 2010) to calculate /6S uncorrected pairwise genetic distances between the new species described herein and other species of Chironius, and compared those distances with recently published data (Sudré et al. 2024).
Species Concept
In this work we follow the unified species concept (de Queiroz 1998, 2007). We infer the existence of the new species described below based on morphological and phylogenetic criteria, which we interpret as evidence of lineage separation.
Results Systematic Account
Chironius whipala sp. nov. urn:|sid:zoobank.org:act:03E2618F-FAAB-48EB-9AAA-8E516511A040
Proposed common English names: Whipala Sipos, Whipala snakes
Proposed common Spanish names: Sipos Whipala, serpientes Whipala
Holotype. MHNC-R 3099 (Fig. 1), an adult male collected in Chaquisacha, Carrasco National Park, Cochabamba Department, Bolivia (17°24’24.42”S, 65°15’34.88”W, 1,337 m) on 21 February 2021 by Rene Carpio-Real at 2345 h in humid montane forests (Yungas).
Paratypes (12). Cochabamba Department: Same collection data as holotype: MHNC-R 3100, juvenile male collected by Oliver Quinteros-Mufioz on 15 March 2021 at 2330 h; MHNC-R 3101, adult female collected by Rene Carpio-Real on 17 December 2020 at 2300 h; MHNC-R 3102, adult male (specimen in poor condition,
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A New Species of Sipo Snake, Chironius
. oe A
Stays
Fig. 1. Male holotype (MHNC-R 3099) of Chironius whipala sp. nov. in preservative. Left: dorsal (top) and ventral (bottom) views of body. Right: dorsal (top), lateral (middle), and ventral (bottom) views of head. Scale bar (head only) = 10 mm. Photographs by Rene Carpio-Real.
found dead), collected by José Balderrama on 27 September 2020; MHNC-R 3133, adult male collected by Oliver Quinteros-Mufioz on 17 April 2021 at 1930 h; MHNC-R 3134, juvenile female collected by Rene Carpio-Real on 20 April 2021 at 2349 h; MHNC-R 3135 juvenile female collected by Rene Carpio-Real on 17 May 2021 at 2152 h; MHNC-R 3227, an adult male found dead in poor condition, collected by Jorge Espinoza on 17 December 2022 at 1500 h near La Payjcha close to type locality, 2,000 m; MHNC-R 3229, an adult male found dead, collected by Bladimir Marca on 25 May 2023 at 1146 hnearArepucho close to type locality, 1,270 m. Santa Cruz Department: MNKR 3073, adult male collected by Walter Romero on 28 October 2001 in Pampagrande, Florida Province, 18°6’0.02”S, 64°6’0.00”’W, 1,300 m; MNKR 3589, adult male collected by Pedro Maida on 18 December 2003 in La Hoyada, Florida Province, 17°55’12.02”S, 64°7712.01°W, 1,730 m; MNKR 4833 adult male and MNKR 4834 adult female, collected by Lucindo Gonzales, Rutty Rodriguez, and Oswaldo Helmig on 15 November 2009 in Laja Tocos, Vallegrande Province, 18°29°41.37°S, 63°43’55.34’W, 1,300 m.
Etymology. The specific name “whipala”’ comes from the original Aymara language, which means “emblem,” i.e., the emblem of the original people of the Andes of Bolivia and an emblem that honors and symbolizes respect for our Pachamama (Mother Earth). According to an anonymous Aymara phrase, “where there 1s a wiphala, love and respect for Mother Earth (Pachamama) and the universe will be represented.”
Diagnosis. Chironius whipala can be distinguished from other species of Chironius by the following combination
Amphib. Reptile Conserv.
of characters: (1) dorsal scale formula 10-10-8 in males, 10-10-10 in females; (2) apical pits absent; (3) paravertebral keels present, inconspicuous in females; (4) ventrals 149-151 in males, 151-155 in females; (5) subcaudals 117—122 in males, 119-120 in females; (6) cloacal plate single; (7) loreal slightly longer than high; (8) maxillary teeth 32; (9) juveniles emerald green or olive green, without markings; (10) adults emerald green, unmarked; (11) black postocular stripe absent; (12) snout yellow; (13) ventrals and subcaudals yellowish or greenish, immaculate; (14) hemipenis short (i.e., ~2X as long as wide), cylindrical, unilobed, with undivided sulcus spermaticus and base covered with tiny spines.
Chironius whipala differs from all known species of Chironius except C. fuscus and C. leucometapus in having 10 dorsal scale rows at midbody, an entire cloacal plate, lightly colored lower portion of supralabials, and keeled paravertebral rows. From C. fuscus (character states in parentheses), C. whipala can be distinguished by having a yellow snout and forehead (head uniformly colored) and lacking both a postocular stripe (present) and enlarged spines on the proximal aspect of the hemipenial body (large spines present) (Dixon et al. 1993; Torres-Carvajal et al. 2019a). From C. /eucometapus (character states in parentheses), C. whipala can be distinguished by having a yellow snout patch covering rostral, first supralabials, and anterior portion of nasals and internasals (rusty brown or coppery orange snout patch covering rostral, first pair of supralabials, internasals, prefrontals, nasals, frontal, and anterior half of supraoculars). Finally, C. whipala differs from both C. fuscus and C. leucometapus in having a much shorter hemipenis (~2X versus > 5X as long as wide) and in lacking apical pits on the dorsal scales.
Description of holotype. Adult male (Fig. 1), total length (TTL) 1,356 mm, SVL 960 mm, tail incomplete; head well differentiated from neck, narrow anteriorly, wider in temporal region, HW 43.6% of HL; snout rounded in dorsal and lateral views, SL 11.4 mm; eye large (EL 6.5 mm), pupil round, EN 20.8% of HL; rostral large, wider than high, visible from above; nasal divided, with large nostril separating anterior half from posterior half; internasals quadrangular, as wide as long, smaller than prefrontals, laterally in contact with nasals; prefrontals slightly wider than long, larger than internasals, each laterally in contact with nasal, loreal, and preocular; frontal bell-shaped, longer than wide, twice the length of suture between prefrontals; parietals large, 1.4 times as long as wide, interparietal suture length similar to length of frontal; postcephalic scales four; preocular single, separated from frontal by prefrontal-supraocular contact; loreal slightly longer than high, in contact with nasal, prefrontal, preocular, and supralabials II and III; postoculars two on each side; temporals 1 + 1; supralabials nine, I in contact with nasal, II in contact with nasal and loreal, III in contact with loreal and
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preocular, IV - VI in contact with orbit (VI also in contact with postocular), VI and VUI in contact with anterior temporal, and IX in contact with both anterior and posterior temporals; infralabials 10/9, 6/5 in contact with chinshields; chinshields in two pairs, anterior pair shorter than posterior pair, both pairs in contact medially, except for last half of posterior pair; maxillary teeth 32; dorsal scales in 10-10-8 rows, smooth without apical pits; ventrals 151; cloacal plate single.
Color of holotype in life. Dorsal background uniformly emerald green, with grey skin between scales; snout yellow; lower portion of supralabials yellowish cream; ventral aspect of head immaculate white; dorsal coloration extending onto lateral tips of ventrals; ventral coloration white anteriorly, yellowish green on first third of body, turning into green tones posteriorly.
Color of holotype in preservative. Dorsum uniformly bluish-green, darker on posterior two-thirds of body; dorsal aspect of head dark brown; snout light brown; lower portion of supralabials and ventral aspect of head cream; venter bluish grey, lighter anteriorly (Fig. 1).
Hemipenis. The hemipenial description is based on the left organ of MHNC-R 3229, an adult male with SVL = 810 mm (Fig. 2). Organ relatively short, ~2X as long as wide, unilobed, non-capitate, and subcylindrical in shape; sulcus spermaticus undivided, linear, running centripetally from base to apex and terminating near center of apex, bordered by naked tissue proximally, spines centrally, and papillae distally; base of organ covered with small spines; basal naked pocket on medial region; proximal half of body densely covered with large spines; distal half of body densely covered with small spines (less than half the size of proximal spines) proximally, which are replaced by papillate fringes and calyces with papillate borders distally; apex with nude area.
Fig. 2. Left hemipenis of Chironius whipala sp. nov. (MHNC-R 3229) in asulcate (left) and sulcate (right) views. Scale bar = 5 mm. Photographs by Diego A. Paucar.
Amphib. Reptile Conserv.
Ai ge : ld fOSO 5 | it Ti al A Fig. 3. General view of Chironius whipala sp. nov. in life. (A) juvenile male, paratype MHNC-R 3100; (B) adult male, para- type MHNC-R 3133; (C) juvenile female, paratype MHNC-R 3135; (D) adult female, paratype MNKR 4834. Photographs by Oliver Quinteros-Mufioz (A, B, C) and Lucindo Gonzales (D).
-68.000 -64.000 -60.000 -56.000 ; at a he | PR cea = aay as Se —=—
~68.000 ; : “64.000 ~ -60.000
Fig. 4. Known distribution of Chironius whipala sp. nov. in Bolivia. The star corresponds to the type locality, and the green line depicts the boundaries of Carrasco National Park.
Variation. Intraspecific variations of Chironius whipala sp. nov. in scutellation and meristic characters are presented in Table 1. While males present reduction in the number of dorsal scales (10-10-8), females maintain the same number along the body (10-10-10). All specimens are similar in color pattern