Nevado de Toluca: habitat for Romerolagus diazi?

The volcano rabbit (Romerolagus diazi), also known as teporingo or zacatuche, is a small rabbit that is endemic to Mexico. In this study we characterized its potential habitat in the Area of Protection of Flora, and Fauna Nevado de Toluca, Mexico. Between April 2016 and November 2017, we sampled 1,807 units to determine the presence of this species using indirect evidence. We found dung pellets that could be attributed to R. diazi in 41 (2.27 %) of the sampled units. In 10 % of these units, we set up camera traps to confirm the presence of the species. Sites with presumed R. diazi pellets were characterised by rocky terrain, with Pinus hartwegii as the dominant tree species, and Festuca tolucensis as the dominant grass. Overall herbaceous cover was over 70 %. Sites observed to have a negative effect on the presence of the pellets were areas with livestock grazing and induced burning. The results of camera trapping did not reveal the presence of R. diazi in Nevado de Toluca.


Introduction
The diversity of hares and rabbits (Order Lagomorpha, Family Leporidae) is high in Mexico, with 15 species. Seven of these are endemic (Farías, 2011). The volcano rabbit, (Romerolagus diazi Ferrari-Pérez 1893), also known as zacatuche or teporingo, is a small, monospecific rabbit found only in the mountains around the southern part of Mexico City. It lives in small groups in runways among grass tussocks, typical of its distribution area. The volcano rabbit is considered endangered (SEMARNAT, 2010;IUCN, 2017). It is threatened by the loss and fragmentation of its habitat (Hoth et al., 1987;Velázquez et al., 2011), introduced species (dogs and cats), hunting, and more recently, climate change (López et al., 1996;Anderson et al., 2009). The main causes for the loss of habitat are anthropogenic fires, livestock grazing, and logging (López et al., 1996).
The volcano rabbit is known to occur along the central Trans-Mexican Volcanic System (TMVS) in discontinuous patches in four volcanoes (Popocatépetl, Iztaccíhuatl, El Pelado and Tláloc) that cover approximately 386 km 2 (Velázquez, 1994). Although there is no historical evidence of sightings of the species, some researchers (Velázquez et al., 1996a) have argued that the rabbit could potentially be found in areas of similar habitat in other volcanoes and mountains in the TMVS such as the Nevado de Toluca, Nevado de Colima, Volcán Tancítaro, Cofre de Perote and Pico de Orizaba. However, credible evidence of their presence in these sites is lacking (Velázquez et al., 1996a;Olascoaga et al., 2015).
In 1975, a volcano rabbit was found in the Nevado de Toluca, in Central Mexico, about 80 km west of Mexico City and near the city of Toluca. It is now deposited in the Instituto Politécnico Nacional Museum (Galindo, C., pers. comm.). There is also anecdotal information regarding sightings in the 1980s, but no evidence was found during a field study in 1987 (Hoth et al., 1987). In 1998, Ceballos et al. (1998) reported finding dung pellets attributable to the species but no further details were given. On the basis of these previous uncertain findings, we attempted to confirm the presence of the volcano rabbit in the Protected Area of Flora and Fauna Nevado de Toluca, focussing on sites with environmental characteristics that are similar to those in its area of distribution.

Sampling
Between 22 April 2016 and 05 July 2017 we searched for the presence of the volcano rabbit in pine-grassland habitat in the PAFFNT ( fig. 1). We established 1807 sampling units (SU) above 3,000 m (Dauber, 1995) situated along every 100 m (3,100, 3,200, etc). Each SU consisted of a circular plot of 0.1 ha (17.86 m radius) to guarantee a minimum sampling intensity (recommended 0.44 % for 12,924 ha (Dauber, 1995).
Two axes were drawn in the north-south and east-west directions crossing the central point of the SU. Within each SU, we searched for rabbit pellets. Two other lagomorph species are found in the area (Sylvilagus floridanus and S. cunicularius). Rabbit pellets found were photographed to determine whether they corresponded to R. diazi (Aranda, 2012). Using this indirect approach, we analysed each photographic record digitally (with ImageJ software) to verify the dorso-ventrally compressed spherical shape and maximum diameter of 10 mm that is typical for the species (fig. 2).

Statistical analysis
We analysed the data by dividing the SUs into two groups: one with presence of pellets attributable to R. diazi (41 sites) and another without pellets (1,766 sites). To identify the variables that correctly classified the SU with and without pellets, we performed a stepwise discriminant analysis, with the quantitati-ve variables. We tested the independence of each qualitative variable and the presence of pellets, with contingency tables and x 2 -tests.

Camera trapping
In 10 % of the sites with pellets attributable to R. diazi, a camera trap was placed in front of latrines to obtain photographic records of the species using the latrine. The cameras were operated day and night for 6 months (July to December, 2017). The presence of different vertebrate species and their relative abundance was obtained using the following indices: where: C is the number of photographic events and SE, the sampling effort (number of cameras per day of monitoring; Maffei et al., 2002;Jenks et al., 2011;Monroy-Vilchis et al., 2011).
The 41 SUs with pellets attributable to R. diazi were found between 3,305 and 3,874 m, with a higher proportion between 3,500 and 3,700 m ( fig. 2)

Discussion
The presence of the volcano rabbit in the Nevado de Toluca has not been confirmed in any scientific study although pellets considered attributable to the species have been reported (Ceballos et al., 1998). In our study, pellets attributable to the volcano rabbit were mostly associated with rocky outcrops, where P. hartwegii was the dominant arboreal species, F. tolucensis was the dominant herbaceous species and the percentage of herbaceous cover was > 70 %. These characteristics are similar to those reported in the Pelado volcano, Mountain Tlaloc and Iztaccíhuatl, where the species is known to exist (Velázquez et al., 1996a(Velázquez et al., , 1996bHunter and Cresswell, 2015).
The PAFFNT is a relatively small area. It is subject to anthropogenic pressure such as agriculture, livestock grazing, logging and feral species (dogs and cats;CONANP, 2016). However, in our study, SUs with pellets were found in habitat considered optimal for the species since they show environmental characteristics similar to other sites of confirmed distribution (Velázquez, 1996).
The presence of the volcano rabbit is related to several factors. The most positive factor is the herbaceous stratum (Fa et al., 1992;Velázquez et al., 1996b) that not only provides protection and refuge from predators (Trigo et al., 2003) but also contributes to diet. Several negative factors have also been described, particularly livestock grazing, controlled burning, and feral dogs (Weber, 2010;García-Aguilar, 2012). In a study conducted in the Iztaccíhuatl volcano, the species was most abundant in the habitat with the highest percentage of grassland and least abundant in areas with more hunting and grazing (Hunter and Cresswell, 2015). This coincides with our results, since the sites with pellets attributed to R. diazi were found in areas with over 70 % of herbaceous cover, mainly Festuca.
The incidence of forest fires within the area is another threat to their ecosystems. In the APFFNT 80 % of fires are intentional (PROBOSQUE, 2012). The main type of fire during the last ten years was the superficial or creeping type (CONANP, 2016), whose most severe damage is reflected on the herbaceous cover. In other distribution sites of R. diazi, however, it has been reported that controlled burning has a significant positive effect on the appearance of the rabbit (Hunter and Cresswell, 2015). In the PAFFNT, however, the incidence of recent fires is not related to the presence of pellets.
Pinus hartwegii grows between 3,000 and 4,000 m on high mountain forests in Mexico (Rzedowski, 2006;Endara et al., 2013). Forests of P. hartwegii are closely related to the presence of pellets attributable to R. diazi. Endara et al. (2013) state that the areas with the greatest deterioration dynamics are Pinus spp. High mountain ecosystems are among the most threatened and impacted ecosystems worldwide due to climate change, which is transforming their landscape, substantially changing environmental conditions, and affecting the viability of biodiversity in these areas (Astudillo-Sánchez et al., 2017).
Camera trapping is a common technique to study medium and large mammals. In rabbits, it has been used to record their presence and calculate their relative abundance (Monroy-Vilchis et al., 2011;Hernández-Hernández et al., 2018). Although we were unable to confirm the presence of R. diazi in the Nevado de Toluca, pellet data suggest it is present the in PAFFNT. Because the evidence is indirect we consider the occurrence of the species should be verified using direct methods such as photographic records. Further photo-trapping should also be carried out in the other sites with ideal habitat characteristics for the species. Ecological and social studies are needed in these sites to gain further knowledge on how to extend the distribution of the species to the Nevado de Toluca.