NATURAL HISTORY AND BIOLOGY OF THE TIANNAN CROCODILE NEWT, TYLOTOTRITON YANGI (URODELA: SALAMANDRIDAE) AT GEJIU, YUNNAN PROVINCE, CHINA WITH ITS CONSERVATION IMPLICATIONS

The natural history of newly discovered species among the genus Tylototriton in South-Eastern Asia is poorly known to date, and most of the species are threatened by extinction. The description of their ecology is crucial to determine their survival status and successful reproduction for conservation purposes. In this study, we assessed the microhabitat preferences of Tylototriton yangi at the type locality, Gejiu, Yunnan province, China, with the Visual Encounter Survey method and by using new important materials. The main habitat is located within a humid subtropical climate and is composed of a large karstic mountain containing some patch of secondary mixed forests and plantations such as tomato fields, pepper and cabbage that are surrounded by scrubs, grasses and small ponds. We provided a complete diagnosis with new morphological data analysing ten adult individuals from the type series to establish a formal description which is absent from the first original description. We compared our results with other related taxa of the genus Tylototriton to updated key determinants between similar species. Thus, we analysed the population structure and the behaviour of T. yangi during a period of ten years, 2008–2017, to assess its survival status and to determine the main threat factors. A total of 186 specimens were found during the whole study predicting a small population inhabiting the type locality, Gejiu. Consequently, T. yangi is now regarded as a highly threatened species that need more attention of Chinese authorities and we suggest regarding it as Endangered due to its small distribution range, the low number of adult specimens observed and ongoing exploitation.


Introduction
Newts and Salamanders belong to the Urodela group. This large group shows a high biodiversity on Earth (Dubois & Raffaëlli, 2009Raffaëlli, 2013;Frost, 2017;AmphibiaWeb, 2018). In the tropical-subtropical region, many new species were recently identified, and some families show an important endemism restricted to small habitats with specific ecological conditions (Hernandez, 2015(Hernandez, , 2016b. In this case, one of the most interesting examples is the Salamandridae family that includes 119 species and occurs in the temperate regions of the northern hemisphere, but also in the tropical-subtropical region in southeast Asia (Duellman & Trueb, 1986;AmphibiaWeb, 2018). This family appears broadly diversified in South-Eastern Asia, where five genera appear (Fei & Ye, 2016;Hernandez et al., 2017). In the tropical region, cryptic popula-tions were found these last decades resulting in the description of several new species. It is observed especially within both the genera Echinotriton and Tylototriton, which form, together with the western Mediterranean Pleurodeles, the tribe Pleurodelini, a primitive lineage within the Salamandridae (Dubois & Raffaëlli, 2009Raffaëlli, , 2012Kurabayashi et al., 2012;Nishikawa et al., 2014Nishikawa et al., , 2015Marjanović & Witzmann, 2015;Hernandez, 2016b,c,d,e). These urodeles are widely distributed from India to Indochina and South-Eastern China, with fossil specimens having been discovered here that are related to the first Urodela in the world (Wake & Özeti, 1969). They are one of the few lineages of sala-They are one of the few lineages of salamanders that appear in the tropical regions, with a poorly known ecology and natural history . Tylototriton and Echinotriton comprise more than 27 species with many undescribed taxa, and most of them are endangered pri-marily because of the habitat destruction in South-Eastern Asia (Seglie et al., 2003;Phimmachak et al., 2015;Hernandez, 2015Hernandez, , 2016bQian et al., 2017). During the last decade, new populations were identified and described in the Province of Yunnan, China, where an important evolutionary radiation occurred resulting from the Pleistocene glaciation among the «Tylototriton verrucosus» group, known as the subgenus Tylototriton Yu et al., 2013). In 2009, the second author found at the pet market of Chengdu city many specimens sold as «Tylototriton verrucosus» for 6 to 10 euros that originated from southern Yunnan province without exact localities. In 2012, these specimens were localised around Gejiu city, Honghe Hani, and Yi Autonomous Prefecture, Yunnan province, China and then studied and described as a new valid species, Tylototriton yangi Hou, Zhang, Zhou, Li & Lü, 2012(Fei et al., 2006Nishikawa et al., 2015;Fei & Ye, 2016). In the past, this new species was reported and identified as «T. verrucosus» from eastern Yunnan province (Fei, 1996(Fei, , 1999. The Tiannan Crocodile newt, Tylototriton yangi is a terrestrial salamander and endemic Chinese species. It inhabits the Karst Mountains including patch of secondary mixed forests with scattered water sources at relatively high elevations of 1600 to 1800 m a.s.l. Fei et al., 2012;Fei & Ye, 2016;Hernandez, 2016e). The species occurs at Gejiu in Honghe county, Honghe prefecture, Yunnan province, China, which is the type locality. Other localities known are distributed in Honghe and Wenshan Counties Fei et al., 2012;Fei & Ye, 2016). For this species, Fei et al. (2012), Raffaëlli (2013) and Sparreboom (2014) added an account in their books, and later we provided new data including a short diagnosis, some ecological and behavioural traits for a better understanding in a book on the genus Tylototriton (Hernandez, 2016e). Nishikawa et al. (2015) confirmed its taxonomic and phylogenetic position within Tylototriton and replaced the taxon T. daweishanensis Zhao, Rao, Liu, Li & Yuan, 2012 as a junior synonym of T. yangi. Wang et al. (2017) studied the reproductive biology in captivity and explained the declining factors for this new species. New studies improved the short description for this species imported for the pet trade in mass. To date, limited information is known for T. yangi regarding its ecology and its microhabitat preferences since the original description (Hernandez, 2016e;Fei & Ye, 2016;Wang et al., 2017). More-over, because the known distribution range of T. yangi overlaps greatly with the major tin-mining sites in southern China, the species is now threatened by extinction. Actually, its current status is not evaluated per IUCN criteria and the species is not protected in China (Hernandez, 2015;IUCN, 2017). In this alarming situation, we present here new major data regarding its ecology with a description of its biology to provide its requirements for conservation purposes.

Climate
The climate at Gejiu site is characterised by relatively high temperatures and evenly distributed precipitation throughout the year (Fig. 2). The Köppen Climate Classification subtype for this climate is «Cfa» Humid Subtropical Climate. According to WorldClim database (version 1.4), the average temperature for the whole year is 15.5°C for the last decade. On average, the warmest month is June with an average temperature of 19.9°C. The coolest month on average is January, with an average temperature of 8.9°C. The average amount of precipitation per year in Gejiu is 1308.1 mm. The month with the most precipitation on average is July with 251.5 mm of precipitation. The month with the least precipitation on average is January with an average of 20.3 mm. There is an average of 164.2 days of precipitation, with the most precipitation occurring in August with 23.9 days and the least precipitation occurring in February with 6.3 days (Fig. 3). In summer, these regions are largely under the influence of moist, maritime airflow from the western side of the subtropical anticyclonic cells over low-latitude ocean waters (Zhu, 1997;Zhu et al., 2005). Temperatures are high and can lead to warm, oppressive nights when the salamanders are active for reproduction. Summers are usually somewhat wetter than winters, with much of the rainfall coming from convectional thunderstorm activity; tropical cyclones also enhance warm-season rainfall in some regions. The coldest month is usually quite mild, although frosts are not uncommon, and winter precipitation is derived primarily from frontal cyclones along the polar front. Finally, our locality is characterised by a «humid subtropical climate» (Kottek et al., 2006, Baker et al., 2010. This climate is warm and temperate (Zhu et al., 2005). When compared with winter, the summers have much more rainfall. The average annual temperature is 17.2°C in Gejiu. About 1139 mm of precipitation falls annually. In summer, these regions are largely under the influence of moist, maritime airflow from the western side of the subtropical anticyclonic cells over low-latitude ocean waters. Temperatures are high and can lead to warm, oppressive nights. These parameters are important factors for the reproduction of the species as suggested for other salamandrid species (Hernandez, 2017).

Field survey
We prospected at the same site during various months of the year (November and May to August) during a ten-year period (2008−2017). The Visual Encounter Survey method (Hayer et al., 1994) was used for observing the crocodile newts. This method is cost effective and does not disturb the habitats of the target species (Ghimire & Shah, 2014). Observations of the vegetation and habitat of the salamanders were carried out by conducting field trips both on sunny and on rainy days. Daily fieldwork was done approximately from 8:00 a.m. to 10:30 p.m., but sometimes later in the evening. For microhabitat study we surveyed almost all types of habitat found in the study area. These include permanent and temporary streams and ponds (including artificial reservoirs and irrigation canals) by means of dip-netting; and surrounding terrestrial habitats, removing stumps, stones and litter Microhabitat characteristics were recorded by collecting data regarding land type, water and soil temperature and type, weather condition, vegetation structure. Temperatures were recorded by using a mercury thermometer and photographs of the vegetation types using a digital camera (Sony Nex-5; Sony Ltd., Japan) throughout the   whole period. Co-ordinates of each locality were collected in situ using a Global Positioning System (Garmin Montana 680; Garmin Ltd., Olahe, KS, USA) and located on maps. Chemical water parameters were measured in situ using a Expresstech @ LCD PH Medidor Digital (Expresstech; Kingpow Company Limited; Hong-Kong; China) for pH. Climate and elevational data were obtained from the WorldClim database (version 1.4).

Morphology
Specimens studied for morphological description are derivated from Hou et al. (2012) and were collected by Hou Mian and Zhou Jia-Jun at Gejiu, Yunnan province, southern China (23°21′35.8″N, 103°09′59.0″E) at 2039 m a.s.l. in the summer periods of 2007-2008. They were fixed in 95% alcohol after preserving muscle tissues in 95% alcohol. Adult specimens were later transferred to 75% alcohol. The ten specimens used for the original description are numbered from NO.SYNU-HM20070801-NT001 to NO.SYNU-HM20070801-NT010. They were deposited at Shenyang Normal University, Shenyang, China. A dial caliper was used (Louisware LSW-CL1810, Louisware Ltd., USA) for measurements to the nearest 0.1 mm. Examined specimens are listed in Table 1. Data for comparisons were obtained from description made by Fei et al. (2006Fei et al. ( , 2012.

General characteristics.
A total of 186 specimens were found during the ten-year period 2008-2017.
Morphometric characteristics. We analysed only ten specimens of the type series to study the morphology of the species and to establish a stable diagnosis. Data for T. shanjing Nussbaum, Brodie & Yang, 1995, T. taliangensis Liu, 1950, T. kweichowensis Fang & Chang, 1932 were obtained from Fei et al. (2006Fei et al. ( , 2012 and Fei & Ye (2016) for comparison with our target species (Table 1).
Holotype   Paratypes. No. SYNU-HM20070801-NT003 to SYNU-HM20070801-NT010, four adult males and four adult females. Collection data are the same as those of the holotype.
Analysis. All adult individuals are morphologically similar to the original diagnosis by Hou et al. (2012) and the descriptions from Fei et al. (2006Fei et al. ( , 2012, Fei & Ye (2016) and Hernandez (2016e). The females are larger and more massive than the males, growing to 172 mm max. in TL, whereas males will measure only up to 158 mm (Fig. 4B).
Diagnosis. The snout is rounded, with pronounced cephalic edges (similar to T. kweichowensis), the vertebral line is prominent, the 15-16 dorsolateral glandular warts are clearly distinct, and the skin is distinctly warty. Both the upper and lower parts are black, and orange pigment is found on the parotoids from where a wide dorsal stripe continues to the tail, on the glandular warts, tips of fingers and toes, the cloaca, and the lateral and ventral parts of the tail.
Comparisons. T. yangi differs from all other known species by having a dark to black colouration on head, dorsum and venter; and a bright orange colouration on corners of the mouth, on dorsolateral glandular warts, parotoids, tail, fingers, toes and the cloacal region. Tylototriton yangi differs from T. kweichowensis by having separate and pronounced dorsolateral glandular warts. Tylototriton yangi differs from T. shanjing by having a black head, a large cranial forma-  tion (cephalic edges) and four black limbs. Tylototriton yangi differs from T. taliangensis and T. verrucosus Anderson, 1871 by having large cephalic edges, a bright red (orange) colouration on dorsolateral glands and tail. Tylototriton yangi differs from the subgenera Yaotriton and Liangshantriton by having a bright reddish to orange colouration on corners of the mouth, vertebral tubercular ridge, dorsolateral glandular warts well pronounced, large parotoids and tail.
Larvae. Larvae were collected at the same site in August 2008 by Hou Mian (Fig. 6). The larvae can be diagnosed as follow: Head flat, labial fold slender, costal groove next to invisibility. Dorsal fin beginning from posterior of the head, along with growth falls back on middle dorsal in gradual. The whole colouration is from light yellow change to tannish-gray or lilac in growth, have irregularly dark spot change to yellow or bluish-gray pattern on the whole length in growth. The colour changes deep. The gill disappears gradually during the growth. Some white spots present to hindbrain and lateral line will raise and the colour changes slowly, reaching to parotid and dorsolateral glandular warts. The spots on the tail will expand onto the whole tail by colouration change in reddish or orange.
Microhabitat composition and characteristics. The vegetation type is one of the key components of the Amphibians microhabitat because most of the species are dependent of the vegetation cover near water sources (Qian et al., 2007). Composition and characteristics of five different structural microhabitats of T. yangi are provided in Tables 2 and 3. Several plant species were identified within the 35 permanent quadrats: trees (2.15%), shrubs (44.08%), herbs (31.19%), and grasses (22.58%). Although specimens of T. yangi were found in the same habitat they have different microhabitat preferences and which varies during different parts of the day and the year, especially during the breeding period. In April up to the beginning of September, animals were found in ephemeral ponds and irrigated canals (Figs. 7 and 8). Ponds were semitransparent with a high concentration of phytoplankton and zooplankton. The soil was primarily silty clay and sandy loam. Thus, five microhabitats were analysed and classified corresponding to the species ecological preferences especially during the breeding period: 1) ephemeral ponds and water bodies with a total of 79 specimens found; 2) irrigation canals with 66 specimens; 3) soils and rich humus composed with fallen leaves with 21 specimens found mostly during the dry season; 4) amount of rocks with 15 specimens; 5) cultivated lands with only five specimens found including juveniles. These microhabitats were full of different species of trees, shrubs, herbs and grasses near to human habitation were identified as important to this salamander (Table 3).     Habitat preferences. We found all of the specimens analysed in this study at Gejiu (Figs. 1 and 7). Xishuangbanna area seems to be a good place for T. yangi but no individuals were observed, only another sister species such as T. shanjing in new southern localities . The habitat of Gejiu is located in a karstic mountain containing some patch of secondary mixed forests and plantations such as tomato fields, pepper and cabbage that are surrounded by scrubs, grasses and small ponds available for reproduction. Here, it shelters in leaf litter composed of large fallen leaves near the pools where humidity levels are very high (77.3-97.6%). The studied site is located at high altitude with highly mineralised red and yellow soils including mainly sandy loam soils in large ponds and silty clay and several ephemeral water bodies. The air temperature was 20.9-22.4°C with a relative humidity of about 86% in July. The larvae were observed in six small ponds fed by slow-flowing streams and co-harbouring mol-lusks, fishes, tadpoles and crabs such as the following species: Babina pleuraden (Boulenger, 1904) Table  3). The water is mainly acidic in all the water bodies analysed with an average of 6.7.
Behaviour. The species is poorly active during the dry and winter period with only eight juveniles found during the whole study (October to March). It is a terrestrial species which is mainly adapted to the underground lifestyle. Adult specimens are fossorial such as T. kweichowensis and are living underground as previously reported during dry and cold periods (Hernandez, 2016e). The present study also shows that these Amphibians were more active in the early hours of the day (9.30-11.00 a.m.) and during the beginning of the nighttime (6.30-11.30 p.m.). Most of the specimens found of T. yangi need a suitable microhabitat within their small terrestrial and aquatic ecosystems for survival purposes. Ephemeral water ponds and canals are crucial for its conservation. At Gejiu's site, the microenvironment which is important for the survival of this threatened species depends on many complex factors (Table 3): i) The presence of water sources and large to small ephemeral ponds highly vegetated with aquatic plants; ii) The nearest presence of shrubs, large rocks, pieces of mixed forests on clay loam soils.
iii) The presence of a various microfauna within the phyla Arthopoda and Mollusca.
Consequently, T. yangi remains almost inactive during the whole winter. Only some juveniles were active on the soil surface probably to find food resources. Its activity is highly associated with rhythm of precipitation and humidity especially between May up to September -October corresponding to its reproductive period (Fig. 3).