The rediscovery of Perret’s toad, Amietophrynus perreti (Schiøtz, 1963) after more than 40 years, with comments on the species’ phylogenetic placement and conservation status

Perret’s toad, Amietophrynus perreti, has not been seen since 1970 and thus believed to be lost. We searched for the species 50 years after its original description and successfully recorded its continued presence at the type locality, where it seems to maintain a viable population. We failed however, to record the species at suitable sites elsewhere and A. perreti could thus indeed be a micro-endemic species, specialized and restricted to the granite inselbergs of the Idanre Hills, Nigeria. We recorded and discuss potential threats and suggest keeping the toad’s current conservation status as ‘Vulnerable’. We investigated the systematic status of Perret’s toad using a mitochondrial fragment of the 16S rRNA gene and could confirm that it is a member of the genus Amietophrynus despite its aberrant larval biology, different to the rest of the genus. In spite of this biological difference, A. perreti is not a phylogenetically isolated lineage, but is nested within a clade of western African Amietophrynus species, such as A. maculatus, A. regularis, A. latifrons and A. togoensis and is sister to the widespread and savannah dwelling A. maculatus.

Since its description the species has rarely been mentioned in the literature. Based on osteological characters (Martin 1972) and acoustic criteria (Tandy and Keith 1972) it was placed in a monotypic group within the genus Bufo. Mills Tandy studied the three toads A. perreti, A. maculatus and A. regularis in 1964 and 1965, occurring in sympatry at the A. perreti type locality, and observed that they differ in size, advertisement call parameters, reproductive season and biology, as well as in microhabitat selection (Tandy and Keith 1972). Based on karyology (2n= 20; Bogart 1968), but without genetic data, Frost et al. (2006) transferred B. perreti into their new genus Amietophrynus, and Channing et al. (2012) redescribed the tadpoles. Schiøtz and Tandy (2004) and Stuart et al. (2008) give the conservation status of the species as 'Vulnerable'.
However, only Mills Tandy seems to have collected new data from the field (Tandy and Keith 1972) and until now, the species is still only known from its type locality. On the webpage of the Amphibian Survival Alliance (ASA 2014) Amietophrynus perreti is listed as being lost, the last observation dating to 1970. We do not know from which data the later date stems. In 2013, 50 years after its original description, we decided to search for the species. Our aims were fourfold: i) document if the species still exists at the type locality, ii) collect data on the population status, iii) search in neighbouring areas for further populations, and iv) investigate the systematic position of this species.

Material and methods
All field work was carried out by two persons (ABO and a field assistant) in the Idanre Hills, Ondo Province, south-western Nigeria (approximately 7°06'N, 5°06'E; see Appendix). The region was visited three times; the first visit from 10-12 October 2013 was only used to get familiar with the topography and to select a suitable field assistant. The second visit took place at the beginning of the dry season (26-28 October 2013). During three days and two nights we applied visual and acoustic encounter surveys (compare Rödel and Ernst 2004) at three sites (see below). Day surveys were conducted at all sites while night surveys were conducted at sites A and C only. The third visit comprised one day and one night survey at sites A and B, respectively, and took place from 13-14 March 2014, the ending of the dry season (Table 1).
Site A (7°06'37.3"N, 5°06'24.1"E) comprised approximately 2 ha situated between two huge rocks, only about 50 m from the town of Idanre (Fig. 2a, c). Water flowed in two valleys between these rocks. The vegetation consisted mainly of shrubs and small trees. Site B (7°06'14.2"N, 5°04'41.3"E), covering about 1.5 ha, was approximately 250 m height on the rocks. This area was relatively flat with patches of vegetation, shrubs and trees, growing on the rocks. Water was flowing down from higher locations, forming rock-pools in some depressions (Fig. 2b). Site C (7°06'32.9"N, 5°04'28.0"E) was part of a cocoa farm adjacent to a large inselberg, including a creek flowing through the farm (Fig. 2e). The creek had a rocky bottom and was 6-11 cm deep. Along the water course, there were few grasses and shrubs.
In addition, we surveyed two further sites with similar habitats (rocky inselbergs). Ikere was about 28 km from Idanre. Two areas were surveyed here on 3-4 November 2013 and 16 March 2014. Site 1 comprised giant rocks and was surveyed for 6 and 4 hours per day and night, respectively (3 day and 2 night surveys, each 2 hours length). With the same sampling effort we investigated site 2, a cocoa plantation near rocks. The second area was situated about 6 km from Akure and was searched on 6 November 2013 for 3.5 hours during the day and night, respectively. A map of all surveyed sites is provided in Figure 3.
All amphibians encountered were recorded. A few vouchers were collected to ascertain species determination. They were euthanized in a chlorobutanol solution and preserved in 75% ethanol. Vouchers are deposited at the zoological collection of A.B. Onadeko at the University of Lagos (no accession numbers) and at the Museum für Naturkunde Berlin (see Appendix for ZMB and GenBank   (Ronquist et al. 2012). Four independent runs were executed to assess convergence, each for 3 million generations, sampling every 1000 generations. Four chains per run were allowed to sample across the entire general time reversible substitution model space and the first 10% of each run were discarded as burn-in before generating a 50% majority-rule consensus tree from the posterior tree samples. Uncorrected 16S p-distances of A. perreti and included Amietophrynus spp. were calculated using PAUP * 4b10 (Swofford 2003).

Results
Occurrence, abundance and threats. We rediscovered Amietophrynus perreti and observed them in sympatry with A. regularis, Phrynobatrachus spp. and Arthroleptis spp. at the study sites of the Idanre Hills inselbergs. We did not observe any A. maculatus, known to occur here as well (Tandy and Keith 1972 Amietophrynus perreti (Fig. 4) were observed during day and night, mainly on higher parts of the rocks. They were encountered on the hard rock surfaces and among low herbal vegetation on sandy soil between the rocks. During October 2013 we observed a total of 31 A. perreti, 16 during the day (12 person-hours search time, Table 1) and 15 during the night (6 p-h). In March 2014 we recorded a total of 12 A. perreti, 5 during day (11 p-h) and 7 (10 p-h) during night time (Table 2). Compared to the other anuran taxa, A. perreti had the lowest abundances.
We observed two potential threats to the survival of the toads. Most importantly some areas around the sites are being cleared for cocoa plantations, the major cash crop in the region. At higher places on the rocks some crops such as pepper, corn and banana are planted. Another cash making venture is the intense cutting of trees that grow among the rocks.
We could not observe A. perreti in two other localities, namely Akure, 6 km and Ikere, about 28 km from Idanre. Both had similar habitats and topography compared to the A. perreti type locality.
Morphology. We examined three male and four female A. perreti vouchers (see Appendix). The males' snoutvent-lengths (SVL: 39.4-40.2 mm) were within the range Schiøtz (1963) and Tandy and Keith (1972) provided for their specimens. None of the presumed male toads exhibited nuptial pads (dry season and hence no reproductive season). The females (55.3-64.4 mm SVL) slightly exceeded the sizes of the known females (Schiøtz 1963, Tandy andKeith 1972). Small differences to the original description concern the size of the metatarsal tubercles, being quite big and clearly distinct (larger in females than in males), and the presence of small, very flat and indistinct warts on the top of the head (no warts according to original description). All toads had comparatively flat heads, slightly pointed in lateral view (rounded in A. regularis and A. maculatus), almost no webbing between toes (distinct webbing between toes in A. regularis and A. maculatus), no tarsal fold (present in A. regularis and A. maculatus), and a white throat in males (dark in A. regularis and A. maculatus). In the preserved vouchers (some of them very flattened after transportation) the parotid glands ranged from less distinct to slightly more pronounced than in A. maculatus, but they were never as huge and smooth as in A. regularis. All our vouchers had more or less the same dorsal pattern as the holotype (compare Fig. 4). In life they have the olive (to yellowish) tinge Schiøtz (1963) mentioned in his description. All  preserved animals had a much clearer and more contrasting pattern than A. regularis and A. maculatus.
Generic assignment. Tandy and Keith (1972) placed A. perreti in its own, monotypic group, although having the same number of chromosomes as the A. regularis-maculatus-latifrons complex (2n= 20, Bogart 1968). A crossing of a female A. maculatus with a male A. perreti produced some embryos to the larval stage, however none of them reached metamorphosis (Blair 1972) [such crossing worked to gastrula or even larval stage even with American species nowadays placed in different genera, namely Anaxyrus boreas, see Blair 1972].

Discussion
We succeeded in rediscovering Amietophrynus perreti at its type locality, 50 years after its original  description, and 43 years after the last observation, if the date of 1970 is correct (ASA 2014). A welcome byproduct of our investigations was that we could confirm the morphological description of the species by Schiøtz (1963), and more importantly, confirm that it is indeed a member of the genus Amietophrynus. It was however, surprising, and in contrast to the osteological results of Martin (1972) and the biological and acoustical analyses of Tandy and Keith (1972), that A. perreti has no isolated phylogenetic position within the genus, but seems to be most closely related to the savannah dwelling A. maculatus (see Rödel 2000 for details of the biology of this species). The 16S sequence data did not sufficiently resolve a number of nodes in the phylogeny, however, the Amietophrynus crown group was well supported, as was the A. perreti-maculatusregularis-latifrons-togoensis clade. The long time gap since the previous observations and the rediscovery of the toad was not due to particular rarity of the species but can be explained by the fact that nobody else searched for the species. We found the toad being locally quite abundant, although our search time at the beginning and towards the end of the dry season certainly was suboptimal. According to Tandy and Keith (1972) A. perreti reproduces during the core rainy season.
Due to their age and peculiar edaphic and climatic conditions, inselbergs are very special habitats with often unique plant and animal communities (Porembski and Barthlott 2000). Unfortunately, so far they have attracted almost exclusively the attention of geologists and botanists. We only know a few studies either focusing entirely (e.g. McLachlan and Cantrell 1980, McLachlan 1981, Osborne and McLachlan 1985, Köhler and Böhme 1996, Schorr 2003 or partly on amphibians on inselbergs (Rödel 1998). As far as we know, A. perreti is the only African anuran species depending exclusively on this habitat type. Schiøtz and Tandy (2004) speculate that A. perreti may occur in other areas of western Nigeria, offering similar habitats, but we did not record the species in two similar areas in closer vicinity of the type locality, nor was the species recorded by Onadeko and Rödel (2009) from other sites in south-western Nigeria. It thus seems possible that A. perreti is indeed an endemic species of the Idanre Hills.
During the current survey we observed no tadpoles of A. perreti. However, the tadpoles require a constant water film on the rocky surfaces where they graze on algae (Fig.  1), most likely in a situation like figured in Figure 2b. During the dry season, although occasional rains occur, such habitats do not exist. A reason for the absence of A. perreti on other inselbergs might be that the existence of this species, with its very special reproductive biology, requires an environment with high humidity persisting for prolonged periods and some water storing vegetation. Only then it seems possible that a constant water film on the rocks is maintained and provides the tadpoles of A. perreti with enough time to finish metamorphosis. The tadpoles seem to actively avoid deeper water and in-stantly, when forced into water, climb out again (Schiøtz 1963), showing a behavior comparable to some Petropedetes tadpoles (Barej et al. 2010). It thus seems that they will not make use of deeper rock-pools (Fig. 2e) and ultimately depend on the wet rock surfaces (Fig. 1).
We know from other inselbergs in the forest zone of Ivory Coast, that these provide a much hotter and dryer environment compared to the surroundings and thus are home to true savannah species, even within a rainforest matrix (Schorr 2003). The fact that true forest in south-western Nigeria disappeared almost completely (see Onadeko and Rödel 2009), may explain that A. perreti simply cannot survive on other nearby inselbergs because the overall climate is too hot and dry. Already Schiøtz (1963) had failed to record the species in nearby inselbergs, surrounded by savannah vegetation. It is also possible that the mere dimensions of the inselberg landscape near Idanre are simply unequaled elsewhere.
According to the original description (Schiøtz 1963) A. perreti is abundant within its habitat. Arne Schiøtz collected 16 adults in July and August, 10 semi-adults and juveniles in January, July and August and 74 tadpoles in July and August 1961. On our surveys we likewise encountered the species frequently, although probably less frequent than Schiøtz (1963). We guess that the toads are not only reproducing during the rainy season (Tandy and Keith 1972), but that this is also the peek activity period and that the Idanre Hills population is still in good shape. The population thus seems viable, but we do not entirely agree with the judgment of Schiøtz and Tandy (2004) that the species does not face "any serious threats at present" because the "rocky habitat is largely inaccessible and useless to humans". Schiøtz (1963) reported that he found all adults at night on bare rocks. He heard males calling in July 1961 during daytime, the voices coming from "very small patches of grass and low vegetation on the rocks, never from the wooded area" (Schiøtz 1963). Tandy and Keith (1972) report similar observations. It thus seems that the toads, at least during the rainy season, do not need forested areas. However, we believe that the current clearing of larger trees from the area, and the use of smaller patches to grow various crops on the rocks may result in a changing micro-climate and altered hydrology, both potentially affecting the toad's survival, especially during the dry season when humid areas are rarer. In addition, the close proximity to a human settlement and to cash crop plantations may expose the toads to agrochemicals and other potentially harmful pollutants.
As the species could not yet be recorded from any other site, the only known population deserves particular attention. Schiøtz and Tandy (2004) already wrote that the species "is intrinsically at risk because of its restricted range". We thus urge, and agree with Schiøtz and Tandy (2004), for setting up a continuous monitoring program of this population and suggest aiming for a protection status for the Idanre Hills in order to assure the long-term survival of Perret's toad.