Zootaxa 4324 (1): 121–141 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4324.1.7 http://zoobank.org/urn:lsid:zoobank.org:pub:6E545FDA-E061-49D9-BA68-79C2ACABE5F4

The “Cobra-preta” of São Tomé Island, Gulf of Guinea, is a new of Laurenti, 1768 (: )

LUIS M. P. CERÍACO1,2,4, MARIANA P. MARQUES2, ANDREAS SCHMITZ3 & AARON M. BAUER1 1Department of Biology, Villanova University, 800 Lancaster Avenue, Villanova, Pennsylvania 19085-1699, USA 2Departamento de Zoologia e Antropologia (Museu Bocage), Museu Nacional de História Natural e da Ciência, Universidade de Lis- boa, Rua da Escola Politécnica, 58, 1269–102 Lisboa, Portugal. 3Natural History Museum of Geneva, Department of and Ichthyology, C.P. 6434, CH-1211 Geneva 6, Switzerland 4Corresponding author. E-mail: [email protected]

Abstract

The Cobra-Preta (black in Portuguese) of Sao Tomé Island in the Gulf of Guinea has historically been referred to as Naja (Boulengerina) melanoleuca (Squamata: Elapidae). Its presence on the island has been traditionally explained as an introduction from the mainland by Portuguese settlers, supposedly to control the rat population. This explanation has been widely accepted by local authorities and even international conservation agencies. The taxonomic identity of this snake has remained undisputed by all taxonomists who have published about it, with the exception of L. Capocaccia in 1961. Arguments supporting the human introduction hypothesis are weak and are contradicted by historical, morpholog- ical and molecular data. Further, the biogeographic history of the Gulf of Guinea oceanic islands and recent insights on the taxonomic identity and evolutionary history of other taxonomic groups occurring there suggest that the Cobra-Preta, in fact, represents a distinct lineage of the melanoleuca group, endemic to São Tomé. We here describe the Cobra Preta as a new species. The new species differs from N. (B.) melanoleuca, its sister species, by a distinct coloration ventral pattern and the type of contact of the sublingual scales. Data on the toxicology, distribution, ecology, folklore and conservation status of the new species are presented.

Key words: Naja, Boulengerina, , São Tomé & Príncipe, conservation, Naja (Boulengerina) peroescobari sp. nov.

Resumo

A Cobra Preta da Ilha de São Tomé, no Golfo da Guiné, tem sido historicamente considerada como pertencente à espécie Naja (Boulengerina) melanoleuca (Squamata: Elapidae). A sua presença na ilha é tradicionalmente explicada como fruto de um introdução por parte dos colonos Portugueses, para suposto controlo da população de roedores. Esta explicação é amplamente aceite pela autoridades locais, bem como pela agências de conservação internacionais. A identidade taxonó- mica desta serpente manteve-se aceite por todos os taxonomistas que lhe dedicaram estudos, à excepção de L. Capocaccia em 1961. Os argumentos que sustentam a hipótese de introdução humana da espécie são consideravelmente fracos e são mesmo contraditos por dados históricos, morfológicos e moleculares. Para além disso, a história biogeográfica das ilhas oceânicas do Golfo da Guiné e descobertas recentes relativas à identidade taxonómica de outros grupos ali existentes, su- gere que a Cobra-Preta representa de facto um linhagem distinta do grupo melanoleuca, endémica da ilha de São Tomé. Neste artigo descrevemos a Cobra-Preta como uma espécie nova. A nova espécie distingue-se da N. (B.) melanoleuca, a sua espécie irmã, pelo seu padrão de coloração ventral e tipo de contacto entre as escamas sub-linguais. Visto que a Cobra- Preta de São Tomé é a única espécie venenosa que ocorre nas ilhas oceânicas do Golfo da Guiné, apresentamos também dados relativos à sua toxicologia, distribuição, ecologia, folclore e estatuto de conservação.

Palavras chave: Naja, Boulengerina, taxonomia, São Tomé & Príncipe, conservação, Naja (Boulengerina) peroescobari sp. nov.

Accepted by Z. Nagy: 28 Jul. 2017; published: 26 Sept. 2017 121 Introduction

In recent years populations of several vertebrates occurring on the oceanic Gulf of Guinea islands of Príncipe, São Tomé and Annobon and long considered to belong to widespread African species complexes have been recognized as distinct from their continental counterparts and described as full species (e.g. shrews, Ceríaco et al. 2015; skinks, Ceríaco et al. 2016). Further, the known richness and endemism of these islands has been increased through the description of several additional vertebrates, including the Príncipe giant gecko, Hemidactylus principensis (Miller et al. 2012), the Leve-Leve Puddle Frog, Phrynobatrachus leveleve (Uyeda et al. 2007), the Adamastor Skink, Trachylepis adamastor (Ceríaco 2015) and Drewes’ Reed-Frog, Hyperolius drewesi (Bell 2016). Other taxa that were once described as endemic species, but were then sunk into the synonymy of mainland species have either been revived (e.g., the green snake Hapsidophrys principis (Boulenger, 1906) by Jesus et al. 2009) or are currently under review. Knowledge of the biodiversity of these islands is thus rapidly changing and the list of Gulf of Guinea endemics is growing. The Gulf of Guinea islands are oceanic and were never in contact with the African mainland. The estimated dates for their emergence are 31 million years for Principe, 14 million years for São Tomé, and 4.8 million years for Annobon (Lee et al. 1994). The combination of climate and geological events, and the existence of important sea currents that have facilitated the rafting dispersal of several mainland terrestrial taxa (Lee et al. 1994, Measey et al. 2007, Uyeda et al. 2007) have promoted rapid rates of speciation (Melo et al. 2011). As a result, the islands are an important biodiversity hotspot, with a high number of endemic species. Additionally, the islands have among the greatest number of endemic bird species per unit area in the world (Dallimer et al. 2009; Leventis & Olmos 2009). The number of introduced land vertebrate species in these islands is low. Besides some common commensal mammals, such as rats, cats and cattle, the Mona Monkey, Cercopithecus mona (Schreber, 1774), African civet, Civettictis civetta (Schreber, 1776) and Least Weasel, Mustela nivalis Linnaeus, 1766 (the path of arrival of the latter was recently documented by Rodrigues et al. 2017) are known to have been introduced by Portuguese settlers since the discovery of the islands in the late 1400s. In the case of , besides the cosmopolitan Tropical House Gecko, Hemidactylus mabouia (Moreau de Jonnès 1818), very few species are not endemic: the Senegal Skink, Trachylepis affinis (Gray, 1838) arrived recently on Príncipe Island (Ceríaco et al. 2016), whereas the São Tomé population of the Western Hinged Terrapin, Pelusius castaneus (Schweigger, 1812) has recently been shown by mtDNA to be very close to West African populations, also suggesting a recent introduction (Fritz et al. 2011). While these cases are unambiguous, some uncertainties surround the identity and origin of the population of cobras of the Naja Laurenti, 1768 on São Tomé Island. Traditionally identified as Naja melanoleuca Hallowell, 1857, and known locally as “Cobra-preta” (Black Snake), the presence of the snake on the island has been cited by many authors from the nineteenth century onwards (Bocage 1866, 1879, 1886, 1889, 1895, 1905; Greef 1884; Bedriaga 1892; Ferreira 1902; Boulenger 1906; Themido 1941; Manaças 1958, 1973; Capocaccia 1961; Schatti & Loumont 1992; Nill 1993; Hofer 2002; Ceríaco 2015), and it plays a role in the local lore. The “Cobra-preta” is considered to be an introduced species on the island and it is anecdotally stated that it was brought to São Tomé by Portuguese farmers who wanted to protect the cocoa and coffee plantations against rodents (see Anonymous 2008; Machado et al. 2013). The island of São Tomé was discovered in December 1471 by the Portuguese navigators Pêro (also known as Pedro) Escobar and João de Santarém. According to the available historical evidence and contemporary reports, the island had no human population or settlements prior to the arrival of the Portuguese (Henriques 1917; Albuquerque 1989). The settlement of the island was not immediate, as the conditions of the islands were unhealthy and extremely harsh for European settlers, and it was not until the beginning of the 16th century that colonization of the island occurred on a large scale. It was about this time that the first reports on the fauna of the island were made. Valentim Fernandes wrote a report made by the Portuguese navigator Gonçalo Pires, who explored the island in 1506, 35 years after its discovery. This report was only written in 1540, and it is still conserved in the Biblioteca Nacional, Lisbon, Portugal. It has been transcribed and published by several other authors (e.g., Henriques 1917). In this report, Pires and Fernandes mention that “very venomous exist on this island, measuring two cubits [approximately 90 cm] in length, and wide as a man’s arm. They stare at men and do not flee. They are killed in the morning when it is still cold and the fat is extracted from the , although during the day when the snakes are on the trees eating birds, people flee from them because the snake’s bite is deadly, but even if one survives there are high chances of death because the skin, the hair and the nails will fall out. These snakes are black. … Black snakes

122 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. of 5 or 6 palms [approximately 110 and 132 cm respectively] with a white collar on the neck, so venomous that when it bites a man, his eyes will explode out of the head and he will die.” (LMPC translation from the Portuguese original). Another report, by Raimundo José da Cunha Matos (1776–1839), military commander and governor of São Tomé in the late eighteenth and early nineteenth centuries, describes the presence of a black snake that could reach “12 or 15 palms” (264 or 330 cm, respectively) in length, was extremely fast, “shines like a mirror”, and the bite of which caused immediate death to humans (Cunha Matos 1916; see also the transcribed version in Henriques 1917). Despite the colloquial language, both these descriptions clearly portray the São Tomé “Cobra-preta”, contradicting the idea that species was introduced by the Portuguese settlers. At the time of both reports, the island was already under Portuguese control, but the number of settlers and economic activities in the island were extremely low. The first agricultural projects on the island date back to the late 1600s with the introduction of sugar cane plantations in the northeast regions of the island, and only in the late 1700s were cocoa and coffee plantations introduced (Henriques 1917). Other problems also arise from the idea of the Cobra-preta being introduced. Firstly, it attributes to Portuguese settlers and farmers from the 16th, 17th and 18th century an idea of biological control and pest management that is still lacking on the island today (LMPC and MPM pers. obs.). Secondly, it assumes that Portuguese settlers would collect live venomous snakes in mainland and introduce them on the island, when it is known that snakes have been among the most feared and hated in Portuguese culture for centuries (Ceríaco 2012). Thirdly, it also assumes that the Portuguese farmers and owners knowingly introduced one of the most venomous species of African snakes into areas where they lived. There are some counter-arguments, such that Portuguese farmers would not care, as the slaves (initially) and contract workers (from the eighteenth century onwards) would be the ones in possible contact with the snake. But this argument is also extremely debatable, as despite the poor working conditions of the slaves/workers, their lives and health were financially important to the plantation owners. Since its first published records, the “Cobra-preta” has been assigned either to Naja haje Linnaeus, 1758 or Naja melanoleuca Hallowell, 1857. Greef (1884) referred to the São Tomé population as a “dark variety” of N. haje, and Bocage (1866, 1879, 1889) referred to it as N. haje var. nigra, which is a nomen nudum as he did not provide a description, although it is possible that Bocage (1866, 1879, 1889) was, in fact, referring the São Tomé population to Naja nigra (Smith 1838), an African spitting cobra currently known as N. nigricollis Reinhardt, 1843), itself a junior homonym of Naja nigra (Gray 1832), a subjective synonym of Naja naja (Linnaeus, 1758). All subsequent authors have placed the São Tomé population in N. melanoleuca, originally described as a subspecies of Naja haje (Hallowell, 1857) based on specimens from ‘Gaboon’ (= ). The species has an extensive distribution across Africa, from Liberia to southern , and several names have been proposed during the late nineteenth century to mid-twentieth century as closely related species and/or subspecies, namely Aspidelaps bocagii Sauvage, 1884, Naja melanoleuca var. leucostica Fischer, 1889, Naja melanoleuca subfulva Laurent, 1955, and Naja melanoleuca aurata Stucki-Stirn 1979 (Broadley 1968). Currently all these names (with the exception of subfulva) are considered synonyms of N. melanoleuca, despite increasing evidence that some of these forms may represent independently evolving lineages, as N. melanoleuca has been found to be paraphyletic and to represent a species complex (D.G. Broadley pers. comm.; September 2014). The case of N. subfulva is a good example of the state of flux in which the taxonomy of this group remains. The existence of two different “forms” of N. melanoleuca—a savannah form, traditionally identified as subfulva, and a “forest form” usually considered as true melanoleuca—was accepted by Broadley et al. (2003) and Chirio & Lebreton (2007), but Broadley & Baylock (2013 referred to subfulva as a subspecies of melanoleuca, admitting the possibility that it may represent a “good species”, and others have treated subfulva as a full species (Chirio & Ineich 2006; Wallach et al. 2014). The taxonomy and phylogeography of African cobras has been intensively reviewed in recent decades, with three new species being described in the last 14 years—Naja nubiae Wüster & Broadley, 2003, from north-eastern Africa; Naja ashei Wüster & Broadly, 2007, from eastern Africa; and Naja senegalensis Trape, Chirio, Broadley & Wüster, 2009, from West Africa. Previous studies (e.g. Slowinski & Keogh 2000, Nagy et al. 2005, Wüster et al. 2007, Kelly et al. 2009) demonstrated that African elapids of the genus Naja were non-monophyletic with respect to Boulengerina and Paranaja. The latter two genera were therefore, consolidated in Naja, with 26 extant species, 11 in Asia and 15 in Africa (Wallach et al. 2009). Four main clades are recognized, and although initially placed in separate subgenera to avoid medical and public health confusion related to generic name changes (Wallach et al. 2009), they have

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 123 subsequently been raised to generic status (Wallach et al. 2014). The species of the Asian lineage were all placed in the subgenus (or genus, according to Wallach et al. 2014) Naja Laurenti, 1768, the non-spitting lineage comprising N. annulifera, N. haje, N. nivea, N. senegalensis, Naja arabica Scortecci, 1932, and Naja anchietae Bocage, 1879, was placed in Uraeus Wagler, 1830, the other non-spitting lineage including the species N. multifasciata, Naja christyi (Boulenger, 1904), N. annulata and N. melanoleuca was placed in Boulengerina Dollo, 1886, and finally all the African spitters were placed in Afronaja Wallach, Wüster & Broadley 2009. Current doubts on the identity of the “Cobra-preta” from São Tomé can have both public health and biodiversity conservation implications. Despite the existence of anecdotal reports regarding the effects of “Cobra- preta” venom, no explicit studies have been conducted, which is particularly concerning as the local population commonly interacts with the snake and the current health services in the country are seemingly unprepared to deal with it. Due to ongoing concerns about the conservation and preservation of the island’s endemic fauna (and attempted eradication of introduced biota), the popular idea that the “Cobra-preta” was introduced by the Portuguese settlers risks the misapplication of conservation measures, possibly opening the possibility of the eradication of this snake. Given the natural history and evolutionary patterns of the island, as well as the historical sources mentioned above, it is not surprising that the São Tomé “Cobra-preta” may represent a different taxon. These suggestions were already made by Capocaccia (1961) after her analysis of the four specimens from São Tomé deposited in the Museo Civico de Storia Naturale “Giacomo Doria” (Genoa, Italy). According to Capocaccia (1961), the presence of “particular characters”, such as the separation of the posterior sublinguals in all of her São Tomé specimens versus the contact of sublinguals in mainland melanoleuca, and the fact that all ventrals were homogenously dark after the 22th, supported the belief that “one day” the São Tomé population would be considered as an “independent geographical race.” Based on the morphological evidence provided by Capocaccia (1961), historical documents that refer to the existence of the snake on the island prior to human settlement, and the general patterns of endemicity on these islands, we investigated the taxonomic status of the “Cobra-preta” of São Tomé. We analyzed the majority of museum specimens available of the São Tomé population and compared them with other members of the subgenus Boulengerina. We also reviewed the nomenclatural history of the group in order to exclude the possibility of the applicability of an existing available name that could apply to the São Tomé population. A fragment of 16S mtDNA was sequenced in order to compare it to available sequences from other species within the genus. Based on our findings, we consider the São Tomé “Cobra-preta” to represent a distinct taxon and describe it here as new.

Materials and methods

Specimens collected for this study were preserved in 10% neutral buffered formalin in the field and later transferred to 70% ethanol. Liver tissue was removed before formalin fixation and preserved in 95% ethanol. For mensural and meristic comparisons we also examined specimens of all four currently recognized species in the subgenus Boulengerina, with special focus on N. melanoleuca throughout its distribution, and including the extant syntypes of Naja melanoleuca Hallowell, 1857. This includes material deposited in Museu Nacional de História Natural e da Ciência (MUHNAC), Lisbon (Portugal); Instituto de Investigação Científica Tropical (IICT), Lisbon (Portugal); Muséum d’Histoire Naturelle (MHNG), Genève (Switzerland); Zoologische Staatssammlung München (ZSM), Munich (Germany), Zoological Museum of the University of Hamburg (ZMH), Hamburg (Germany); the Museum für Naturkunde (ZMB), Berlin (Germany); the Academy of Natural Sciences of Drexel University (ANSP), Philadelphia (USA); the American Museum of Natural History (AMNH), New York (USA); the Museum of Vertebrate Zoology (MVZ), University of California, Berkeley (USA); and the Museum of Comparative Zoology (MCZ), Harvard University, Cambridge (USA) (see Appendix 1). These records include the majority of the specimens of São Tomé population available in world museums. The material studied by Capocaccia (1961) was not examined directly, but we incorporated her data into our study. We employed standard measures and scale counts used in other works on the genus Naja (Wüster & Broadley 2003; Broadley & Wüster 2004; Wüster & Broadley 2007; Trape et al. 2009) in order to be able to compare our data with those in the published literature. Sixteen characters related to scalation and color pattern were recorded from a series of 15 specimens of “Cobra-preta” from São Tomé, as well as 64 other N. melanoleuca from the

124 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. African mainland, including the four original ANSP syntypes. Whenever possible, for each specimen the following characters were recorded: sex; snout–vent length (SVL); tail length (TL); number of ventrals following Dowling’s count; number of subcaudals (excluding the cloacal scale); number of preoculars; number of suboculars; number of postoculars; number of anterior temporals; number of posttemporals; number of supralabials; number of infralabials; neck scale rows (NSR); midbody scales rows (MSR); ventral scale rows (VSR); proportion and distribution of white ventral scales; as well as general coloration pattern. For molecular comparisons we sequenced partial fragments of the mitochondrial 16S gene from the São Tomé specimens collected, and compared these with available 16S sequences for the genus Naja on GenBank. Salt DNA extraction protocols were performed on two São Tomé specimens and three Naja melanoleuca specimens, using a lysis solution of Proteinase K, 10% sodium dodecyl sulfate, RnaseA, and extraction buffer (1 M Tris, 5 M NaCL, 0.25 M EDTA, and distilled water). A 5 M NaCl solution was used to extract DNA and was subsequently washed with ethanol. DNA extractions are held in a -20˚C freezer for future use. A fragment of the mitochondrial 16S gene was amplified using the primers 16Sa (5‘ CGCCTGTTTATCAAAAACAT 3’) and 16Sb (5’ CCGGTCTGAACTCAGATCACGT 3’) from Simon et al. (1994). Polymerase chain reaction (PCT; 25 µl) consisted of 1x PCR Buffer (20 mM Tris-HCl, 50 mM KCl), 3.0 mM MgCl2, 0.4 uM primer, 200 uM each dNTP, 1.25 units of Taq polymerase, and approximately 25 ng of extracted DNA. Thermal cycling was as follows: initial denaturation step: 5 min at 85 °C; 35 cycles: denaturation 35 s at 94 °C, primer annealing for 3 s at 52 °C; extension for 1 min at 72 °C; and a final step of 5 min at 72°C. PCR products were sent out for clean-up and sequencing by GeneWiz (New Jersey, USA). Sequences were aligned using the ClustalW alignment tool in MEGA 6.06 (Tamura et al. 2013). We encountered no issues during the alignment of the newly generated sequences with those available from GenBank. We performed a Maximum Likelihood (ML) analysis on the 16S dataset using the RaxML HPC 7.4.2. (Stamatakis 2006) implemented in raxmlGUI 1.3 (Silvestro & Michalak 2012). Optimum substitution models were selected using ModelTest 3.7 (Posada & Crandall 1998) using the Akaike informative criterion. Phylogenetic robustness was estimated in the ML analyses by running 1000 random rapid addition bootstrap replicates (Felsenstein 1985). We regarded tree topologies with bootstrap values (bs) of 70% or greater as well supported (Huelsenbeck & Hillis 1993). Pairwise comparisons of uncorrected sequence divergences (p-distance) were computed in MEGA 6.06 (Tamura et al. 2013).

Results

Morphology. Morphologically, there are several characters that distinguish the São Tomé form from the mainland population, such as differences in size, lepidosis, scale counts and color patterns. The São Tomé population is, on average, larger (SVL mean = 125 cm; min = 79 cm, max = 218 cm; n= 12) than the majority of the analyzed mainland material (SVL mean = 113 cm; min = 93 cm, max = 192; n= 47). However, none of the examined São Tomé specimens reached the maximum length of 310 cm (total length) reported from a specimen from by Villiers (1965). There is a consistent difference in the contact of the posterior chin shields between the São Tomé population and the continental population, as in the former these scales are never in contact due to the intrusion of a large scale between them, whereas in the latter these are always in contact, either across their full extent or at least anteriorly (Fig. 1). This difference was already mentioned by Capocaccia (1961) and was found to be constant in all specimens examined by us. Coloration. The distinct pattern in the São Tomé population, with no specimen presenting white scales posterior to the 22th ventral scale, contrasts with the extensive (yet variable) distribution of white ventral scales in mainland melanoleuca. The characteristic throat coloration pattern for N. melanoleuca has been considered a clear distinctive character of the species by numerous authors including Schmidt (1923), Villiers (1950a), Laurent (1956), Trape & Mané (2006) and Pauwels & Vande (2008). Ventral color pattern is also one of the main characters that differentiate the nominotypical form of N. melanoleuca from subfulva. Described by Laurent (1956), as a “race” of melanoleuca, the latter taxon replaces the nominotypical form (typical from forest areas) in more open savanna areas, and it can be distinguished from melanoleuca through a combination of coloration and morphological characters: 1) a brown dorsal coloration (black in melanoleuca); 2) venter commonly whitish, sometimes spotted in adults, or forming a gradient from whitish to blackish that usually starts near the 50th ventral and extends posteriorly about 25 to 75 ventral scales (very dark coloration starts abruptly at approximately the 50th

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 125 FIGURE 1. Comparison of the ventral scales between Naja (Boulengerina) peroescobari sp. nov. (A—holotype: MB03- 001065) and Naja (Boulengerina) melanoleuca (B—syntype: ANSP 6878). Red color denotes the posterior sublinguals, while grey color denote the intrusive gular scale. ventral of melanoleuca); 3) a general lack of dark pectoral bands; 4) a lower number of ventrals (203–211, mean = 207.4, among 18 male subfulva versus 211–221, mean = 216.2, in 12 male melanoleuca; 208–218, mean = 213.0, in 21 female subfulva female versus 210–226, mean = 221.8, in 5 female melanoleuca); 5) a longer anterior temporal scale (sometimes divided), usually almost as long, or sometimes as long as a subocular (considerably smaller in melanoleuca); 6) the presence of three posterior temporals in the majority of specimens versus two in melanoleuca; 7) and the type of contact between the posterior temporals, which is short in subfulva and long in melanoleuca (Laurent 1956). Besides the morphological characters, as the case of the number and dimensions of the temporals (FitzSimons 1962; Broadley 1983, 1990) coloration has chiefly been used to differentiate melanoleuca from subfulva. Villiers (1950b, 1965) already mentioned the variable coloration patterns of the melanoleuca group (clearly including subfulva in his concept of melanoleuca), and discussing Naja melanoleuca from Southern Africa FitzSimons (1962) [as well as Broadley 1983 and 1990] mentioned “Colour: Above, head (and sometimes also neck) brown, and body and tail shiny black throughout, or anterior half to two-thirds of body light to dark brown with flecking or spotting of black, passing to uniform shiny black posteriorly, with or without scattered white spots or black annuli. Below, creamy white to yellowish white, uniform, speckled or blotched in varying degree with black. Some, or all of labials pale, i.e. white to yellowish white, with black edging.”. Broadley et al. (2003), referring to the Zambian form of melanoleuca—considered by them as “N. m. subfulva Laurent 1956,” mentioned regarding its coloration that the species “are brown to blackish-brown above, often passing to glossy black towards the tail. The belly is yellow heavily speckled with black. The labials (lips) are yellow, edged with black. In other parts of the range, there are different colour phases. Those from forested regions of central Africa are glossy black, with black and white ventral banding anteriorly.” In a more recent publication, Broadley & Baylock (2013), based on “accumulating molecular data” suggested that subfulva “is a good species, with a huge, but fragmented, range extending from southern Ethiopia and Somalia south to KwaZulu-Natal in South Africa, and

126 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. west to the Niger delta and Angola,” and its coloration is “Yellowish-brown above, heavily blotched with black, more dense posteriorly, often becoming uniform black on the tail; underside bright yellow, heavily blotched with black. Juveniles may have up to five black bands on the throat, but these fade out in adults.” Just as between melanoleuca and subfulva, the ventral coloration pattern appears to be a constant character that separates the São Tomé population from both true melanoleuca and subfulva. In general, the São Tomé population has a unique white ventral collar, composed by approximately 10 to 22 predominantly white scales on the anterior part of the throat (or two smaller white collars composed by 6 to 8 scales each) and a homogeneous dark venter from there posteriorly (including the cloacal scales and subcaudals; see Figure 2); in contrast to the extensively anteriorly banded yellow/ white and black venter of most West African melanoleuca (Schmidt 1923; Villiers 1950a; Trape & Mané 2006; Pauwels & Vande 2008), as well as to the completely yellow/cream ventrals heavily speckled with black or brown of subfulva (Laurent 1956; Broadley et al. 2003; Broadley & Blaylock 2013), or the Central and East African forest fringe populations which have a cream or white belly with broad black crossbars and blotches throughout the venter (e.g., Schmidt 1923: “In two specimens the venter is mottled with light color to the hundred and first and one hundred and thirty-fifth ventral.”). None of the analyzed specimens from São Tomé had any white scales from the 23rd scale posteriorly, whereas in the continental population the all of the specimens examined had these white scales a until at least the 50th ventral.

FIGURE 2. Distribution of white color on the first 200 ventral scales of São Tomé “Cobra-preta” (n= 12; top) and continental populations of Naja (Boulengerina) melanoleuca (n= 47; down).

Molecular. Molecular analyses with the mitochondrial gene 16S reveal that the São Tomé population is nested within the subgenus Boulengerina and sister to West African N. melanoleuca (Fig. 3), from which it presents a 2.2 to 2.4% divergence for the same marker (Table 1). This divergence comparable to some interspecific divergences between other valid species in the genus using the same marker (Table 1). Our results also suggest that subfulva is a distinct species (Fig. 3). Our tree recovers a very similar topology to the majority of phylogenetic studies on cobras (Wüster et al. 2007; Wallach et al. 2009; Trape et al. 2009). Systematics. Our results show that the São Tomé population of “Cobra preta” can be clearly distinguished from other N. melanoleuca in a number of features of color pattern, scalation and mtDNA sequence data. Given the congruent patterns of variation in morphology and mtDNA, we consider our data to provide evidence that this form represents a different species. As noted in the introduction, despite the existing evidence that N. melanoleuca is a species complex, the taxonomy and nomenclature of the group has mostly been stable for decades. The complex was being reviewed by Wolfgang Wüster and Donald G. Broadley (1932–2016), until the recent death of the latter put a tragic temporary halt to this important revisionary work. Very few names are currently in the synonymy of N.

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 127 melanoleuca, and none of these names appear to be applicable to the São Tomé form (we consider names coined by Raymond Hoser to be unavailable; see Kaiser et al. 2013, Kaiser 2014, and Measey 2013, although none would apply here in any case). No previously described cobra has its type locality on São Tomé, and all existing names, in particular melanoleuca Hallowell, 1857 (type locality: Gabon), bocagei Sauvage, 1884 (type locality: “Gabon” & “Majumba” in the ), subfulva Laurent, 1955 (type locality: “Lwiro” and “Kivu”, Democratic Republic of the Congo), leucostica Fischer, 1885 (type locality: “Cameroon”, “Ogowé” and “Gaboon”) and aurata Stucki-Stirn 1979 (type locality: “From Bamenda northwards as far as Wum and We including the plains north of Kishong” [Cameroon]), are applicable to sets of populations of cobras that were represented in our morphological and/or molecular analyses, and were shown to be highly distinct from this island form. Since no name is available for the São Tomé population, we describe it as new:

TABLE 1. Species and specimens used for mtDNA analysis and corresponding GenBank accession number, locality and voucher and sample size.

Species GenBank accession no. Locality and voucher (if any) Sample size Naja (Boulengerina) peroescobari MF497425 São Tomé Island [MB03-000985; MB03-001065] 2 sp. nov. Naja (Boulengerina) annulata AY188049.1 Zaire (from Pet Trade) 1 Naja (Boulengerina) melanoleuca MF974426; MF974427 Ghana, Volta Region [MVZ245386]; 2 Ghana, Volta Region [MVZ249816]. Naja (Boulengerina) subfulva JF357949.1; MF974428 unknown; Mozambique, Zambezia Province 2 [MVZ266146] Naja (Afronaja) mossambica GQ359744.1 Marromeu, Mozambique 1 Naja (Afronaja) pallida GQ359745.1 Tanzania 1 Naja (Afronaja) nubiae GQ359746.1 Unknown 1 Naja (Afronaja) katiensis GQ359743.1 Doussoudiana, Mali 1 Naja (Afronaja) ashei GQ359742.1 Watamu, Kenya 1 Naja (Afronaja) nigricollis GQ359754.1 Lara, Kaélé, Cameroon 1 Naja (Uraeus) arabica GQ359749.1 Saudi Arabia 1 Naja (Uraeus) annulifera GQ359753.1 Zimbabwe 1 Naja (Uraeus) nivea EU624272.1; GQ359755.1 South Africa; ? 2 Naja (Naja) naja L10674.1 Asia 1

FIGURE 3. Maximum Likelihood tree for Naja, based on the available 16S sequences. Values on the nodes represent bootstrap support. Labels read as “Species [GenBank Accession number—Country]”.

128 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL.

Naja naja

5.0% Naja katiensis

1.8% 5.7% Naja nigricollis

2.2% 1.6% 4.5% Naja mossambica

1.0% 2.0% 1.8% 4.5% Naja ashei

4.3% 4.5% 4.9% 4.1% 7.2% Naja pallida

2.5% 4.4% 3.8% 4.8% 3.7% 6.5% Naja nubiae

3.8% 4.1% 4.1% 4.3% 4.7% 3.9% 6.0% Naja arabica

0.8% 3.7% 3.9% 3.9% 4.1% 4.5% 3.7% 6.7% Naja annulifera

– – – – – – – – 1.9 2.2% 1.5 1.7% 3.6 3.9% 4.0 4.4% 4.3 4.9% 4.6 5.1% 5.1 5.8% 4.0 4.6% 6.5% Naja nivea

– 4.8 5.1% 4.4% 4.6% 3.9% 3.9% 3.6% 3.3% 3.9% 2.9% 5.5% Naja annulata

– – – – – – – – – – – 2.3 2.7% 3.5 4.4% 3.4 3.7% 3.4 3.5% 2.8 3.6% 3.2 4.7% 3.9 4.1% 3.4 3.9% 3.8 4.3% 2.7 2.9% 5.8 6.2% Naja subfulva

4.9% 4.9% – 3.1% 4.5% 4.8% 5.2% 4.1% 3.9% 4.3% 3.9% – – – – – – – – 2.4 2.5% 4.6% 4.3 4.2% 4.6 5.0 3.9 3.7 4.1 3.7 5.5% Naja melanoleuca

2.4% 2.9% 3.1% 6.5% 5.1% 4.9% 5.2% 5.1% 4.5% 4.3% 4.9% 3.9% 6.0%

– – – – – – – – – – – – – 2.2 2.5 2.7 5.1 4.7 4.5 4.9 4.7 4.1 3.9 4.5 3.5 5.5 Naja peroescobari

Uncorrected distancespairwise between different the taxa.

TABLE 2. Naja peroescobari Naja melanoleuca Naja subfulva Naja annulata Naja nivea Naja annulifera Naja arabica Naja nubiae Naja pallida Naja ashei Naja mossambica Naja nigricollis Naja katiensis Naja naja

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 129 Naja (Boulengerina) peroescobari sp. nov. Figs 4–5

Naja haje (Greef 1884: 47; Bocage 1886: 69; Bocage 1895a: 24) Naja haje var. nigra (Bocage 1866: 51; 1879: 87; 1889: 34; Vieira 1886: 237) Naja haje var. melanoleuca (Bedriaga 1982: 299, 440) Naja melanoleuca (Bocage 1905: 94; Manaças 1973: 228; Nill 1993: 71; Hofer 2002: 78, 89; Schatti & Loumont 1992: 31; Themido 1941: 6) Naja melanoleuca melanoleuca (Capocaccia 1961: 299; Manaças 1958: 190) Naia melanoleuca. (Boulenger 1906: 215; Ferreira 1902: 133)

Holotype. MUHNAC/MB03–001065 (Fig. 4), adult male collected in the vicinity of Praia Inhame (0.028636º N, 6.523203º E, WGS-84; 17 m above sea level), São Tomé Island, Republic of São Tomé e Príncipe, by Luis Ceríaco, Mariana Marques and Ana Carolina Sousa on 24 February 2016. The specimen was chopped in half by a local man with a machete, and collected minutes after this event. The choice of this particular specimen as holotype is justified as it is the source of the comparative molecular data. Paratypes. All specimens from the Island of São Tomé, Republic of São Tomé and Príncipe. Eight specimens: IICT 18-1972, adult male collected in Ribeira Peixe (0.090278º N, 6.615278º E, this and all following coordinates use map datum WGS-84; 17 m above sea level) by an unknown collector on 24 December 1972; IICT 20-1967, adult female collected in Santa Josefina (0.247882º N, 6.738315º E; 69 m above sea level) by an unknown collector on October 1967; IICT 2-1966, adult female, without specific locality (São Tomé island) by an unknown collector on 20 February 1966; ZMH R10526, unsexed adult, without specific locality (São Tomé island) collected by Carl Weiss in the late 1840s; ZMH R10527, unsexed adult, without specific locality (São Tomé island) collected by Richard Greef in 1879/1880; MHNG 2518.41 and MHNG 2518.42, both unsexed adults collected in Rio Angra Toldo (0.156881 N, 6.668792º E; 75 m above sea level) by Tilman Nill in 3 April 1991; MHNG 2462.43, adult male collected in the environs of Neves (0.353192 N, 6.634972º E; 281 m above sea level) by Heft and Fahr on 8 August 1989. Diagnosis. Naja peroescobari is placed in the subgenus Boulengerina (as defined by Wallach et al. 2009) on the basis of having 2–4 maxillary teeth, the penultimate supralabial height reaching eye level the combination of one preocular and one anterior temporal, rostral much broader than deep, internasals shorter than the prefrontals, dorsal scales smooth and fangs not modified for spitting. Morphologically, N. peroescobari differs from other members of the subgenus Boulengerina by a combination of coloration and scalation patterns. It differs from N. (Boulengerina) chrysti in having 19 dorsal scale rows (17 in chrysti), three infralabials contacting the chinshields (four in chrysti), and by its homogeneous dark coloration (brownish in chrysti). Naja (Boulengerina) chrysti is considered to be strictly aquatic (Chippaux 2006), whereas N. peroescobari is mainly terrestrial. It also differs from N. (Boulengerina) annulata by the lower number of dorsal scales (19 in peroescobari versus 21 to 25 in annulata), and by its coloration, as annulata has between 21–23 dark bands (the first 3–5 simple, subsequent bands double) over a yellow or brown dorsum (contrasting with the homogeneous black of peroescobari), and the ventral coloration (uniformly dark brown in annulata, versus white banded throat, followed by homogeneous black of peroescobari). Naja peroescobari differs from N. (Boulengerina) multifasciata in its size (260 cm maximum in peroescobari versus maximum total length reported for multifasciata 76 cm; Chippaux 2006), by having two postoculars (three in multifasciata), in having 19 dorsal scale rows (15–17 in multifasciata), a higher number of subcaudals (30 to 39 in multifasciata versus 52 to 70 in peroescobari), a higher number of ventral (153 to 175 in multifasciata versus 208 to 215 in peroescobari), and by its coloration (multifasciata is uniformly light yellow on the ventral side, and the dorsal scales are yellow in the anterior part and black in the posterior, with a dark head, separated by the rest of the body by a white collar; Chippaux 2006). Naja peroescobari differs from the more closely related West African N. melanoleuca, with which it was formerly confused, having a combination of white and black bands, but the white bands are always confined to the first 22 ventral scales, whereas in N. melanoleuca these white bands extend throughout the first two thirds of the venter, usually between the 80th to the 100th scale (max. 125th scale, min. 50th scale; in the type series the last white ventral scale of syntype ANSP 6878 corresponds to the 81st scale, the other having it on the 83th (ANSP 6876), 86th (ANSP 6875) and 90th (ANSP 6879). This is even more evident when comparing the new species with N. subvulva, which has whitish scales mottled with dark spots extending all across the venter. Also the dorsal coloration of N.

130 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. peroescobari is uniformly shiny black, never exhibiting the whithish/yellow markings of the dorsal neck area common in N. melanoleuca. In terms of scalation, the most striking difference between N. peroescobari and N. melanoleuca is the contact of the posterior sublinguals. In N. peroescobari the posterior chin shields are mostly never in contact due to the intrusion of a large scale between them, whereas in N. melanoleuca these are always in contact, either fully across their extent or at least in the anterior part (Meirte 1992 considered this character [sous- linguales postérieures se touchent] as the way to differentiate N. melanoleuca in the key to African snakes). These differences were already mentioned by Capocaccia (1961) and are here confirmed with a larger sample of both taxa.

FIGURE 4. Holotype of Naja (Boulengerina) peroescobari sp. nov. (MUHNAC/MB03–001065).

Description of holotype. Body dimensions: Snout-vent length 1700 mm, tail length 380 mm, dorsal head length (snout to end of parietal suture) 46.2 mm, lateral head length (snout to posterior end of lower jaw articulation) 61 mm. Head width across supraocular 24.8 mm, maximum overall width of head 45.5 mm. Head broad, hearth-shaped from above. Eye moderate, diameter half the distance from mouth or from nostril. Body scalation: 208 ventrals, 66 subcaudals, all paired, anal scale entire, the intact tail terminates in a spine. Dorsal scale rows: 24 on the neck, 19 at midbody, 14 one head length ahead of vent. Head scalation: preoculars 1/1, postoculars 3/3, supralabials 7/7 the third and fourth of which contact the orbital rim, infralabials 8/8 of which the first three contact the anterior chin shields. Cuneate scale absent. Anterior temporals 1/1, posterior temporal 2/2. Two temporals and nuchals contact the lateral and posterior edges of parietals. Rostral 1.3 times wider than high, visible from above. Anterior chin shields adjacent to each other, posterior chin shields separated by one elongate scale. Nasal scale entirely divided into a prenasal and a postnasal scale by the large, vertically elongate nostril. Frontal slightly longer than wide (11.7 x 11 mm), longer than distance from rostral (11.4 mm), shorter than supraoculars (14.2 mm), widest along anterior edge; shape pentagonal, anterior edge slightly concave, posterior edge ends in obtuse angle, border with supraoculars straight. Color and pattern: Head uniformly black dorsally, lateral side cream with large black marking on the anterior/posterior borders of the supralabials and infralabials. Underside of head uniformly pearly white. Dorsal color of the total length of body uniformly shiny black. Interstitial skin black. Throat scales and ventral pattern (Fig. 4): first ten scales uniformly white, except the seventh scale, which presents a small dark blotch at the left side of the scale. Eleventh ventral scale black on the half left, and white with small scattered black dots on the right. Twelfth ventral scale uniformly black. Ventrals 13–15 white, except the fourteenth scale, which has a small dark blotch at the left side of the scale. Ventrals 16–17 uniformly black. Ventrals 18–19 white, but the latter with some black intrusion on the posterior edge. The remainder of the ventral and subcaudal

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 131 scales are uniformly black, and the distal lateral edges of these are continuous in color with the lower dorsal scale rows. Variation. Variation in scale counts and body measurements of the paratypes of N. peroescobari are reported in Table 3. Midbody scale rows 19–21; Neck scale rows 21–25; Tail scale rows 13–16. Due to the limited number of N. peroescobari specimens for which sex was confirmed, no sexually dimorphic differences could be found in the subcaudal scales of the species, which range from 52 to 70 (52 to 69 in females, n= 4; 57 to 70 in males, n= 3). The same applies to ventral scales, which range from 208 to 215 (208 to 214 in females; 208 to 211 in males). The posterior sublinguals are always separated by the intrusion of a scale, however in paratype MHNG 2518.42 the intrusion of this scale between the posterior sublinguals is not evident, as in the other specimens, and both scales contact narrowly in the anterior part. All the paratypes have one anterior temporal followed by two posterior temporals. However in MCHNG 31002a, Capocaccia (1961) reported three posterior temporal instead of two. The anal scale is usually entire in all the type series, with the exception of the paratype IICT 18-1972, where it is divided. Supralabials always 7, whereas the number of infralabials varies between 7 and 9. Largest male examined (MUHNAC/MB03–001065, Praia Inhame, São Tomé) 1700 + 380 = 2080 mm; largest female (IICT 20-1967, Santa Josefina, São Tomé) 2180 + 420 = 2600 cm. All specimens examined were homogenous black on the dorsal areas, entirely agreeing with the holotype, while in the ventral pattern, despite small variations on the arrangement of the white scales (see Fig. 2), no specimen had any white scales distal to the 22th ventral. Etymology. The species is named after Pêro (also known as Pedro) Escobar (?–?), one of the 15th-century Portuguese navigators who discovered São Tomé Island on December 21, 1471, Annobon Island on January 1, 1472, and Príncipe island on January 17, 1472, and participated in the discovery of the ocean route from Portugal to India under the command of Vasco da Gama (circa 1460–1524) in 1497, as well as in the expedition of Pedro Álvares Cabral’s (circa 1467–circa 1520) that “discovered” Brazil in 1500. We adopt the Portuguese local common name “Cobra-preta” and suggest the English common name “São Tomé cobra” for this species. Distribution. The species appears to be widely distributed through the island, except the drier savanna areas in the northeast (Fig. 6). Greef (1884) noted that the species was confined to high areas, from 300 to up 900 m in the northeast “Cordilheira”, and that it was much more common on the southeastern part of the island, near Angra de São João (0.1167° N, 6.6500° E; 134 m above sea level), but it has subsequently been found elsewhere: Roça Minho (0.266667º N, 6.633333º E; 712 m above sea level; Bocage 1905), Roça Saudade (0.288319° N, 6.636209° E; 815 m above sea level; Bocage 1905), Água-Izé (0.218727° N, 6.727969° E; 19 m above seal level; Boulenger 1906, Capocaccia 1961), Roça Porto Alegre (0.033333° N, 6.533333° E; 16 m above sea level; Manaças 1958), Roça Santa Josefina (0.247882° N, 6.738315° E; 69 m above sea level; Manaças 1973), “Cascata” and surroundings (0.2854722° N, 6.6254416° E; 929 m above sea level; Nill 1993); Angra Toldo, also referred as Rio Angra Toldo (0.156881° N, 6.668792° E; 75 m above sea level; Nill 1993), Rio Quicha (location unknown; Nill 1993) and Generosa (0.348305° N, 6.551019° E; 81 m above sea level; Nill 1993). Two specimens from the California Academy of Sciences collections (CAS 219402 and CAS 219403) are from Bom Sucesso (0.288394° N, 6.612155° E; 1154 m above sea level) and Morro Esperança (0.299743° N, 6.616667° E; 998 m above sea level), respectively. The species is also known from Roça Bombaim (0.246481° N, 6.632732° E; 465 m above sea level; LMPC & MPM pers. obs.). The type locality represents the southernmost known record of the species, and also the lowest elevation recorded (21 m). The species is not known from the neighboring Rolas Islet.

TABLE 3. Variation in size and scalation characters displayed in São Tomé’s “Cobra preta”, Naja peroescobari sp. nov., and continental Naja melanoleuca. Values are organized as mean (minimum–maximum; standard deviation). Measurments are rounded off to the closest 1 mm. “Cobra preta” (n= 15) Continental Naja melanoleuca (n= 47) Snout-vent length 1250 (790–2180; 40.3) 1105 (370–1920; 46.9) Tail length 280 (110–420; 8.7) 235 (80–435; 10.4) Ventrals 210 (208–215; 2.2) 218 (200–231; 6.4) Sub-caudals 69 (52–70; 5.9) 64 (33–71; 5.6) Neck scale rows 24 (21–25; 1.5) 24 (22–29; 1.9) Midbody scale rows 19 (19–21; 0.7) 19 (16–21; 0.9) Tail scale rows 13 (13–16; 1.3) 13 (12–16; 0.8)

132 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. FIGURE 5. Live picture of Naja (Boulengerina) peroescobari sp nov. from near Santa Josefina. Specimen not collected. Photo by Tiziano Pisoni.

Medical relevance and folklore. There is currently no available information on the venom of this snake. Local people (as well as historical reports) refer to the snake as highly venomous, causing death in a few hours, but no credible reports about the symptomology were recorded during our expeditions to the island (L.M.P.C & M.P.M pers. obs.). Vieira (1886) mentions that the species is highly venomous (even recording the local saying: “homem mordido, homem perdido”, meaning “man bitten, man lost”), but only bites when attacked or stepped on. Local informants claim that when bitten, patients usually cut the afflicted limb off, and there are also reports of people who are on the top of palm trees to extract palm wine who simply toss themselves to the ground to avoid being bitten whenever they encounter a snake. The manuscript report of José Gonçalves Correia (1888–1954), Portuguese naturalist who collected birds in São Tomé for the American Museum of Natural History in 1928/1929, mentions that after a bite, the afflicted area should be burnt with a hot iron in the first 24 hours (Correia 1928). Further studies on the venom and also on the symptomology and bite incident records are needed to understand the potential medical relevance of this species to the human population. A possible island effect in the venom should

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 133 also not be discounted, as snake venom is known to be highly affected by ecological and geographic conditions (Chippaux et al. 1991). In some parts of the island, namely around Roça Bombaim, the species is captured by locals to serve as a local delicacy, and the meat is sold in the capital city market (L.M.P.C & M.P.M pers. obs.). The recent development of political ties between São Tomé and Príncipe and China, and the expected arrival of Chinese workers to the country, is a potential cause of concern, as snakes from the genus Naja are commonly used in the Chinese Traditional Medicine (Zheng & Zhang 2000) and gastronomy (Zhou & Jiang 2005), and conservation problems related to the exploitation of wildlife by Chinese workers have been reported for other parts in Africa (Munson & Ronghui 2012; Bersacola et al. 2014). Habitat and Natural History notes. Bocage (1895) reports a skull of a Least Weasel (Mustela nivalis) in the stomach contents of one specimen. Specimen IICT 18-1972 had an invasive Black rat (Rattus rattus) in its stomach, but knowledge on the trophic ecology of the species is limited. Vieira (1886) mentions that the species prefer shaded and moist habitats. The snake is mostly seen in forested areas in the southern half of the island, being absent in the northeast regions. It is commonly seen on roads basking during the day.

FIGURE 6. Known distribution of Naja (Boulengerina) peroescobari sp. nov. on São Tomé Island represented by red dots, based on bibliographic and museum records. The white-center dot represents the holotype locality.

134 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. HCIB! !#%!#"! HCIB! '#! !$ HCIB! '# !$ aHC! S $!& aHC! S $!% H8CIB! " !q! H8CIB! " !p! H8CIB! " !i! H8CIB! " !h! H8CIB! "&$($! !‡’ƒr!†r vr†!hq!h‰hvyhiyr!†ƒrpv€r†! DD8U! (&! ' DD8U! (%& ! Najaperoescobari DD8U! ! (%%! H7" ('$!  %$!  !!! ! !! ! !! ! !! ! !! ! !! ! "! ! !! ! !! ! !! ! !! ! !! ! !! ! !! ! !! !Hrh†ˆ r€r‡†!hq!†phyh‡v‚!‚s! ! Tƒrpv€r!! H7" !"#$%&'(& U’ƒr!Tr‘!TWG!€€! C‚y‚‡’ƒr! ! &!UG!€€! Ar€hyr!Wr‡ hy†!! ! ! "'! Qh h‡’ƒr! Qh h‡’ƒrTˆiphˆqhy†! !'! Qh h‡’ƒr %(! ! sr€hyr!Q r‚pˆyh †! ! !! ! sr€hyr!Q‚†‡‚pˆyh †!"! ! '! ! ! 4!8ˆrh‡r†! ! "! ! !! 6‡r v‚ ! #!! ! $!!‡r€ƒ‚ hy†! &$! Hhyr! "! ! #!Q‚†‡ ! !"! !Ur€ƒ‚ hy†! &#! €hyr! %(! ! "!Tˆƒ hyhivhy†!&! ! !! !'! !!!Ds hyhivhy†!'!! €hyr! ! ! %&! ! "!ITS! " ! %$! 4! &!! HTS! ! ! $&! ! ! !"! "! &!WTS! ! !#! &! ! !'!! 6hy!Tphyr! (! %(! ! ! '! "! Qh h‡’ƒr ! @‡v r!! "! $!! &! Qh h‡’ƒr ! ! 4!! &! Qh h‡’ƒr ! ! %!! ! &! Qh h‡’ƒr !#! ! "! ! &! Qh h‡’ƒr! ! !'! 4! %'! @‡v r!! !(! !#! '! ! "! &'&! @‡v r! "! &! (! ! $! !(! 4! ! 9v‰vqrq !!!! !! "! '! ! ! ! @‡v r! !! !&%! "! &! 4! !$! %$! @‡v r! "! !(!! (! &!! (! ! @‡v r! "! &! ! &! #! !#! sr€hyr! ! @‡v r!! (! &! ! ! %$! "! %! ! &! ! ! (!! '! %(! ! "! $! !#! &! @‡v r! (!! ! '! @‡v r! ! %! "! !#! &! @‡v r! !! ! @‡v r! '! ! !$! "! &! @‡v r! ! (! ! ! ! '! "! %! &! (! !!! ! '! "! &! (! !! $! '! (! &! ! "! '! ! !

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 135 Discussion

The importance of combining multiple lines of evidence to support taxonomic and evolutionary hypothesis (integrative taxonomy) is currently regarded as a standard in evolutionary biology (Padial et al. 2010). The identity of São Tomé’s Cobra-Preta is supported by molecular, morphological, biogeographic, and also historical data. The placement of N. peroescobari as sister to N. melanoleuca is not surprising, and was already expected based on its morphological resemblance and taxonomic and nomenclatural history. The specific identity of the São Tomé Cobra-Preta was also supported through morphological analyses. While scale counts and size overlap between the new species and mainland N. melanoleuca, certain scalation characteristics and coloration clearly allow its specific identification. The molecular divergence (2.4%) between N. melanoleuca and N. peroescobari for the 16S gene fits within the range of genetic distances across the same gene in the genus (see Table 2). Molecular and morphological results support recognition of the São Tomé cobra as a separate evolutionary lineage, specifically distinct from N. melanoleuca. Similar to the majority of the island endemic terrestrial vertebrates, it is likely that the original stock that colonized São Tomé originated from the West African mainland by rafting through one of the two major rivers that flow into the Gulf of Guinea, the Volta and the Níger, or maybe even the Congo, as supported by evidence from other groups that occur in São Tomé and have their closest relatives on the African mainland (Ptychadena, Measey et al. 2007; Crocidura, Ceríaco et al. 2015; Trachylepis, Ceríaco et al. 2016; Hyperolius, Bell et al. 2015, 2016; Panaspis, Soares et al. in prep.). However, given the limited availability of N. melanoleuca samples from the surrounding areas, as the geographic origin of the cobra cannot be pinpointed as precisely as in the above cited cases. Our results also support the recognition of subfulva as a valid species within the subgenus Boulengerina. This is not surprising, as subfulva was described as a subspecies of melanoleuca and has traditionally being considered a “savanna form” of the latter. The majority of the distinctive characters of the subgenus according to Wallach et al. (2009) are present in subfulva. In the context of a more integrative approach to taxonomy, historical information can provide important clues regarding the identity or origin of certain species. A famous case is that of Fernando de Noronha Island in Brazil, from whence the 1503 reports of the Italian navigator Americo Vespuci (1454–1512) gave important information regarding several endemic taxa, as for example the presence of the Noronha skink, Trachylepis atlantica (Schmidt, 1945), or the extinct endemic species and monotypic genus of rodent Noronhomys vespuccii Carleton & Olson, 1999. The present situation is an example of the importance of integrating historical data on biological and evolutionary studies, as it can provide important and useful starting points or information. Combined with the other types of evidence, the early navigator reports can be crucial in resolving confusion regarding the arrival of animals to island and, in the present case, in falsifying the hypothesis of intentional introduction of the species by humans. This description also highlights the importance and the need of solid taxonomic background when addressing supposedly “invasive” or “introduced” species The anecdotal (but widespread) idea that the São Tomé’s Cobra- preta was an introduced alien species (an idea even conveyed by conservation programs as ECOFAC/RAPAC, see Anonymous 2008), could represent a menace to the species. There are currently several examples of invasive snakes around the world (e.g. Meshaka et al. 2000; Rodda & Savidge 2007; Silva-Rocha et al. 2015). One of the most well-known cases of invasive snake is the Brown Tree Snake, Boiga irregularis, on the island of Guam, where it was accidently introduced after World War II, and has had a serious impact on native fauna (Rodda & Savidge 2007). However, this is clearly not the case of the São Tomé’s Cobra-preta. In fact, our current knowledge on the ecology of N. peroescobari points out that the species preys on the invasive R. rattus and M. nivalis, possibly representing one of the few local predators capable of controlling invasive mammal populations. As for the majority of the herpetofauna of São Tomé and Príncipe, little is known regarding the distribution, ecology, natural history and conservation status of the newly described species. This lack of data is also true for its toxicology and potential human healthcare implications. More dedicated and focused studies are needed to address these issues and contribute to a better understanding of the biology of the spectacular herpetofauna of the country.

Acknowledgments

The authors want to thank several entities and persons who contributed to this study. From São Tomé e Príncipe,

136 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. special thanks is owed to Arlindo Ceita Carvalho, Director of the General Office for the Environment from the Ministry of Environment and Natural Resources who provided collecting and exporting permits for this study. Cristiane Silveira, from MUHNAC and Ishan Agarwal from Villanova University provided valuable laboratory support during the molecular amplification and sequencing. Arianna L. Kuhn provided some extra measurements and photographs from specimens from the AMNH. Carolina Sousa participated in the fieldwork and was responsible for the initial sighting of the holotype. Ishan Agarwal, Bill Branch, Wolfgang Wüster and Eli Greenbaum critically reviewed this manuscript and provided useful and valuable comments and corrections. The authors also wish to thank to the following herpetological collections managers and curators: Ned Gilmore (ANSP), Frank Burbrink & David Kirzirian (AMNH), Carol Spencer (MVZ), José Rosado (MCZ), Michael Franzen (ZSM), Jakob Hallermann (ZMH) and Frank Tillack (ZMB) for the access to their collections. Tiziano Pisoni kindly provided a live photography of the species. This paper is dedicated to Lilia Capocaccia, Italian herpetologist and former director and curator of the amphibians and reptiles collection of the Museo Civico di Storia Naturale “G. Doria” in Genoa (Italy), who firstly proposed a specific identity to the São Tomé “Cobra Preta”. The corresponding author was supported by National Science Foundation funds (DEB 1556255).

References

Albuquerque, L. (1989) Introdução à História dos Descobrimentos Portugueses. Europa-América, Lisboa, 294 pp. Anonymous (2008) Bulletin ECOFAC/RAPAC São Tomé et Príncipe. Compte rendu du mois de Août 2008. Available from: http://www.rapac.org/index.php?option=com_docman&view=download&alias=40-aout-2008&category_slug=bulletin- ecofac-rapac-sao-tome-et-principe&Itemid=105 (accessed 4 July 2016) Bedriaga, J. (1893) Note sur les Amphibiens et Reptiles recueillis par M. Adolphe Moller aux îles de la Guinée. O Instituto, 40, 299–303, 432–440. Bell, R.C., Drewes, R.C., Channing, A., Gvozdik, V., Kielgast, J., Lotters, S., Stuart, B.L. & Zamudio, K.R. (2015) Overseas dispersal of Hyperolius reed frogs from Central Africa to the oceanic islands of São Tomé and Príncipe. Journal of Biogeography, 42 (1), 65–75. https://doi.org/10.1111/jbi.12412 Bell, R.C. (2016) A new species of Hyperolius (Amphibia: Hyperoliidae) from Príncipe Island, Democratic Republic of São Tomé and Príncipe. Herpetologica, 72 (4), 343–351. https://doi.org/10.1655/Herpetologica-D-16-00008.1 Bersacola, E., Svensson, M.S., Bearder, S.K. & Mills, M. (2014) Hunted in Angola. Surveying the Bushmeat Trade. Swara, 38, 31–36. Bocage, J.V.B. (1866) Lista dos reptis das possessões portuguesas d’Africa ocidental que existem no Museu de Lisboa. Jornal das Sciencias Mathematicas, Physicas e Naturaes, 1 (1), 37–56. Bocage, J.V.B. (1879) Subsidios para a Fauna das possessões portuguezas d’Africa occidental. Jornal das Sciencias Mathematicas, Physicas e Naturaes, 7 (26), 85–96. Bocage, J.V.B. (1886) Reptis e Amphibios de S. Thomé. Jornal das Sciencias Mathematicas, Physicas e Naturaes, 11 (42), 65– 70. Bocage, J.V.B. (1889) Breves considerações sobre a fauna de S. Thomé. Jornal das Sciencias Mathematicas, Physicas e Naturaes, Segunda Série, 1 (1), 33–36. Bocage, J.V.B. (1895) A doninha de São Tomé. Jornal das Sciencias Mathematicas, Physicas e Naturaes, Segunda Série, 4 (13), 24–27. Bocage, J.V.B. (1905) Contribution à la Faune des quatre îles du golfe de Guinée (suite). Jornal das Sciencias Mathematicas, Physicas e Naturaes, Segunda Série, 7, 65–96. Boulenger, G.A. (1906) Report on the reptiles collected by the late L. Fea in West Africa. Annali del Museo Civico di Storia Naturale di Genova, 3 (2), 196–216. Broadley, D.G. (1968) A review of the African cobras of the genus Naja (Serpentes: Elapinae). Arnoldia (Rhodesia), 3, 1–14. Broadley, D.G. (1983) FitzSimons’ Snakes of Southern Africa (Revised Edition). Delta Books, Johannesburg, 376 pp. Broadley, D.G. (1990) FitzSimons’ Snakes of Southern Africa (Revised Edition). Jonathan Ball & Ad. Donker Publishers, Parklands, 387 pp., 84 color pls., Addendum. Broadley, D.G., Doria, C.R. & Wigge, J. (2003) Snakes of . An Atlas and Field Guide. Edition Chimaira, Frankfurt am Main, 280 pp. Broadley, D.G. & Blaylock, R. (2013) The Snakes of Zimbabwe and Botswana. Edition Chimaira, Frankfurt am Main, 387 pp. Broadley, D.G. & Wüster, W. (2004) A review of the Southern African ‘non-spitting’ cobras (Serpentes: Elapidae: Naja). African Journal of Herpetology, 53 (2), 101–122. https://doi.org/10.1080/21564574.2004.9635504 Capocaccia, L. (1961) Contributo allo studio dei serpenti delle isole del Golfo di Guinea. Annali del Museo Civico di Storia

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 137 Naturale “Giacomo Doria,” 72, 285–309. Carleton, M.D. & Olson, S.L. (1999) Amerigo Vespucci and the rat of Fernando de Noronha: a new genus and species of Rodentia (Muridae, Sigmodontinae) from a volcanic Island off Brazil’s continental shelf. American Museum Novitates, 3256, 1–58. Ceríaco, L.M.P. (2012) Human attitudes towards herpetofauna: the influence of folklore and negative values on the conservation of amphibians and reptiles in Portugal. Journal of Ethnobiology and Ethnomedicine, 8, 8. https://doi.org/10.1186/1746-4269-8-8 Ceríaco, L.M.P. (2015) Lost in the middle of the sea, found in the back of the shelf: A new giant species of Trachylepis (Squamata: Scincidae) from Tinhosa Grande islet, Gulf of Guinea. Zootaxa, 3973 (3), 511–527. https://doi.org/10.11646/zootaxa.3973.3.6 Ceríaco, L.M.P., Marques, M., Jacquet, F., Nicolas, V., Colyn, M., Denys, C., Sardinha, P. & Bastos-Silveira, C. (2015) Descritption of Crocidura fingui, a new endemic species of shrew (Mammalia, Soricomorpha) from Príncipe Island (Gulf of Guinea). Mammalia, 79 (3), 325–341. https://doi.org/10.1515/mammalia-2014-0056 Ceríaco, L.M.P., Marques, M.P. & Bauer, A.M. (2016) A review of the genus Trachylepis (Sauria: Scincidae) from the Gulf of Guinea, with the descriptions of two new species in the Trachylepis maculilabris (Gray, 1845) species complex. Zootaxa, 4109 (3), 284–314. https://doi.org/10.11646/zootaxa.4109.3.2 Chippaux, J.-P., Williamns, V. & White, J. (1991) Snake venom variability: Methods of study, results and interpretation. Toxicon, 29 (11), 1279–1303. https://doi.org/10.1016/0041-0101(91)90116-9 Chippaux, J.-P. (2006) Les serpents d’Afrique occidentale et centrale. Institut de Recherche pour le Développement (IRD), Paris, 311 pp., 16 pls. Chirio, L. & Ineich, I. (2006) Biogeography of the reptiles of the . African Journal of Herpetology, 55 (1), 23–59. https://doi.org/10.1080/21564574.2006.9635538 Chirio, L. & Lebreton, M. (2007) Atlas des reptiles du Cameroun. Muséum national d’Histoire naturelle/ Institut de Recherche pour le Développement (IRD), Paris, 688 pp. Correia, J.G. (1928) Unpublished field notebook, housed in the Ornithology. Department of the American Museum of Natural History, New York. Dallimer, M., King, T. & Atkinson, R.J. (2009) Pervasive threats within a protected area: conserving the endemic birds of São Tomé, West Africa. Animal Conservation, 12, 209–219. https://doi.org/10.1080/21564574.2006.9635538 Felsenstein, J. (1985) Phylogenies and the Comparative Method. The American Naturalist, 125 (1), 1–15. https://doi.org/10.1086/284325 Ferreira, J.B. (1902) Lista dos Reptis e Batrachios da colecção do Sr. Newton. Jornal das Sciencias Mathematicas, Physicas e Naturaes, Segunda Série, 6, 281–284. FitzSimons, V. (1962) Snakes of Southern Africa. Purnell & Sons, Cape Town, 423 pp. Fritz, U., Branch, W.R., Hofmeyr, M.D., Maran, J., Prokop, H., Schleicher, A., Siroky, P., Stuckas, H., Vargas-Ramírez, M., Vences, M. & Hundsdorfer, A. (2011) Molecular phylogeny of African hinged and helmeted terrapins (Testudines: Pelomedusidae: Pelusios and Pelomedusa). Zoologia Scripta, 40, 115–125. Greef, R. (1884) Ueber die Fauna der Guinea-Inseln S. Thomé und Rolas. Sitzungsberichte der Gesellschaft zur Beforderung der gesammten Naturwissenschaften zu Marburg, 1884 (2), 41–80. Hallowell, E. (1857) Notice on a collection of reptiles from the Gaboon country, West Africa, recently presented to the Academy of Natural Sciences in Philadelphia by Dr. Henry A. Ford. Proceedings of the Academy of Natural Sciences of Philadelphia, 9, 48–72. Henriques, J. (1917) A ilha de S. Tomé sob o ponto de vista histórico-natural e agrícola. Boletim da Sociedade Broteriana, 27, 1–197. Hofer, D. (2002) The São Tomé and Principe Handbook. D. Hofer Verlag, Bern, 152 pp. Huelsenback, J.P. & Hillis, D.M. (1993) Success of phylogenetic methods in the four-taxon case. Systematic Biology, 42, 247– 264. https://doi.org/10.2307/2992463 Jesus, J., Nagy, Z., Branch, W.B., Wink, M., Brehm, A. & Harris, D. J. (2009) Phylogenetic relationships of African green snakes (genera Philothamnus and Hapsidophrys) from São Tomé, Príncipe and Annobon islands based on mtDNA sequences and comments on their colonization and taxonomy. The Herpetological Journal, 19, 41–48. Kaiser, H. (2014) Best practices in herpetological taxonomy: errata and addenda. Herpetological Review, 45, 257–268. Kaiser, H., Crother, B.I., Kelly, C.M.R., Luiselli, L., O’Shea, M., Ota, H., Passos, P., Schleip, W.D. & Wüster, W. (2013) Best practices: In the 21st Century, taxonomic decisions in herpetology are acceptable only when supported by a body of evidence and published via peer-review. Herpetological Review, 44, 8–23. Kelly, C.M.R., Barker, N.P., Villet, M.H. & Broadley, D.G. (2009) Phylogeny, biogeography and classification of the snake superfamily Elapoidea: a rapid radiation in the late Eocene. Cladistics, 25, 38–63.

138 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. Laurent, R. (1955) Diagnoses préliminaires de quelques Serpents venimeux. Revue de Zoologie et Botanique Africaine, 51, 127–139. Laurent, R. (1956) Contribution à l’Herpétologie de la Région des Grands Lacs de l’Afrique centrale. Annales du Musée Royal du Congo Belge, Série in 8°, Sciences Zoologiques, 48, 1–390, 31 pls. Lee, D.C., Halliday, A.N., Fitton, J.G. & Poli, G. (1994) Isotopic variations with distance and time in the volcanic islands of the Cameroon line: evidence for a mantle plume origin. Earth and Planetary Science Letters, 123, 119–138. Leventis, A.P. & Olmos, F. (2009) As aves de São Tomé e Príncipe. Um Guia Fotográfico / The Birds of São Tomé and Príncipe. A Photoguide. Editora Aves e Fotos, São Paulo, 142 pp. Machado, A.M., Sousa, A.F., Marques, R.M. & Pereira, S.M. (2013) Guia Turístico de São Tomé e Príncipe. Pocket Tropics, Évora, 288 pp. Manaças, S. (1958) Anfíbios e Répteis das ilhas de São Tomé e do Príncipe e do Ilhéo das Rolas. In: Anonymous. Conferência Internacional dos Africanistas Ocidentais, Vol IV, Junta Investigação do Ultramar, Lisboa, pp. 179–192. Manaças, S. (1973) Alguns dos anfíbios e répteis da província de S. Tomé e Príncipe. In: Anonymous. Livro de Homenagem ao Prof. Fernando Frade. Junta Investigação do Ultramar, Lisboa, pp. 219–230. Matos, R.J.C. (1916) Corografia história das Ilhas de São Tomé, Príncipe, Ano Bom e Fernando Pó. Imprensa Nacional, Lisboa, 120 pp., 20 pls. Measey, J. (2013) Taxonomic publishing, vandalism and best practice: African Journal of Herpetology makes changes that will safeguard authors. African Herp News, 60, 2–4. Measey, G.J., Vences, M., Drewes, R.C., Chiari, Y., Melo, M. & Bourles, B. (2007) Freshwater paths across the ocean: molecular phylogeny of the frog Ptychadena newtoni gives insights into amphibian colonization of oceanic islands. Journal of Biogeography, 34, 7–20. https://doi.org/10.1111/j.1365-2699.2006.01589.x Melo, M., Warren, B.H. & Jones, P.J. (2011) Rapid parallel evolution of aberrant traits in the diversification of the Gulf of Guinea white-eyes (Aves, Zosteropidae). Molecular Ecology, 20, 4953–4967. https://doi.org/10.1111/j.1365-294X.2011.05099.x Meirte, D. (1992) Clés de determination des serpents d’Afrique. Annales Sciences Zoologiques, 267, 1–152. Meshaka W.E., Loftus, W.F. & Steiner, T. (2000) The herpetofauna of Everglades National Park. Florida Scientist, 63, 84–103. Miller, E.C., Sellas, A.B. & Drewes, R.C. (2012) A new species of Hemidactylus (Squamata: Gekkonidae) from Príncipe Island, Gulf of Guinea, West Africa with comments on the African–Atlantic clade of Hemidactylus geckos. African Journal of Herpetology, 61 (1), 40–57. https://doi.org/10.1080/21564574.2012.666680 Munson, P. & Ronghui, Z. (2012) Feeding the Dragon: Managing Chinese resources acquisition in Africa. Seattle Journal of Environmetal Law, 2 (1), 343–382. Nagy, Z.T., Vidal, N., Vences, M., Branch, W.R., Pauwels, O.S.G., Wink, M. & Joger, U. (2005) Molecular systematics of African Colubroidea (Squamata: Serpentes). In: Huber, B.A., Sinclair, B.J. & Lampe, K.H. (Eds.), African biodiversity: molecules, organisms, ecosystems. Springer Verlag, Bonn, pp. 221–228. https://doi.org/10.1007/0-387-24320-8_20 Nill, T. (1993) Die Schlangen der Insel São Tomé (Golf von Guinea) (Reptilia: Serpentes). Faunistische Abhandlungen Staatliches Museum für Tierkunde Dresden, 19 (7), 71–73. Padial, J., Miralles, A., De la Riva, I. & Vences, M. (2010) The integrative future of taxonomy. Frontiers in Zoology, 7, 16. https://doi.org/10.1186/1742-9994-7-16 Pauwels, O.S.G. & Vande Weghe, J.P. (2008) Reptiles du Gabon. Smithsonian Institution, Washington D.C., 272 pp. Posada, D. & Crandall, K.A. (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics, 14 (9), 817–818. https://doi.org/10.1093/bioinformatics/14.9.817 Rodda, G.H. & Savidge, J.A. (2007) Biology and Impacts of Pacific Island Invasive Species. 2. Boiga irregularis, the Brown Tree Snake (Reptilia: Colubridae). Pacific Science, 61 (3), 307–324. https://doi.org/10.2984/1534-6188(2007)61[307:BAIOPI]2.0.CO;2 Rodrigues, M., Bos, A.R., Schembri, P.J., Lima, R.F., Lymberakis, P., Parpal, L., Cento, M., Ruette, S., Ozkurt, S.O., Santos- Reis, M., Merila, J. & Fernandes, C. (2017) Origin and introduction history of the least weasel (Mustela nivalis) on Mediterranean and Atlantic islands inferred from genetic data. Biological invasions, 19 (1), 399–421. https://doi.org/10.1007/s10530-016-1287-y Schätti, B. & Loumont, C. (1992) Ein Beitrag zur Herpetofauna von São Tomé (Golf von Guinea) (Amphibia et Reptilia). Zoologische Abhandlungen des Staatliches Museum für Tierkunde Dresden, 47 (1), 23–36. Schmidt, K.P. (1923) Contributions to the Herpetology of the Belgian Congo based on the collection of the American Museum Congo Expedition 1909-1915. Part II—Snakes. Bulletin of the American Museum of Natural History, 49, 1–146. Silva-Rocha, I., Salvi, D., Sillero, N., Mateo, J.A. & Carretero, M.A. (2015) Snakes on the Balearic Islands: An Invasion Tale with Implications for Native Biodiversity Conservation. PLoS ONE, 10 (4), e0121026. https://doi.org/10.1371/journal.pone.0121026 Silvestro, D. & Michalak, I. (2012) RaxmlGUL: a graphical front-end for RAxML. Organismal Diversity and Evolution, 12, 335–337. https://doi.org/10.1007/s13127-011-0056-0

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 139 Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H.-M. & Flook, P. (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America, 87, 651–701. https://doi.org/10.1093/aesa/87.6.651 Slowinski, J.B. & Keogh, S. (2000) Phylogenetic relationships of elapid snakes based on cytochrome b mtDNA sequences. Molecular Phylogenetics and Evolution, 15, 157–164. https://doi.org/10.1006/mpev.1999.0725 Stamatakis, A. (2006) RaxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22, 2688–2690. https://doi.org/10.1093/bioinformatics/btl446 Stucki-Stirn, M.C. (1979) Snake Report 721: A Comparative Study of the Herpelogical Fauna of the Former West Cameroon/ Africa, with a Classification and Synopsis of 95 Different Snakes and Descriptions of Some New Sub-species. Herpeto- Verlag, Teuffenthal, 650 pp. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30, 2725–2729. https://doi.org/10.1093/molbev/mst197 Themido, A.A. (1941) Répteis e batráquios das colónias portuguesas. Memórias e Estudos do Museu Zoológico da Universidade de Coimbra, 119, 1–31. Trape, J.-F. & Mané, Y. (2006) Guide des Serpentes d’Afrique Occidentale. IRD Éditions, Paris, 226 pp. Trape, J.-F., Chirio, L., Broadley, D.G. & Wüster, W. (2009) Phylogeography and systematic revision of the (Serpentes: Elapidae Naja haje) species complex, with the description of a new species from West Africa. Zootaxa, 2236, 1–25. Underwood, G. (1979) Classification and distribution of venomous snakes in the world. In: Lee, C.Y. (Ed.), Handbook of Experimental Pharmacology. Springer-Verlag, Berlin, pp. 41–60. https://doi.org/10.1007/978-3-642-66913-2_2 Uyeda, J.C., Drewes, R.C. & Zimkus, B.M. (2007) The California Academy of Sciences Gulf of Guinea Expeditions (2001, 2006) VI. A new species of Phrynobatrachus from the Gulf of Guinea Islands and reanalysis of Phrynobatrachus dispar and P. feae (Anura: Phrynobatrachidae). Proceedings of the California Academy of Sciences, 58, 367–385. Vieira, L. (1886) Noticia ácerca dos productos zoologicos trazidos da ilha de S. Thomé para o Muzeu Zoologico da Universidade de Coimbra pelo Sr. Adolpho F. Moller, em 1885. O Instituto, 34, 235–241. Villiers, A. (1950a) La collection de serpents de L’I.F.A.N. Institut Français d’Afrique Noir, Dakar, 155 pp. Villiers, A. (1950b) Les serpents de l’Ouest Africain. Institut Français d’Afrique Noir, Dakar, 148 pp. Villiers, A. (1965) Les serpents de l’Ouest Africain. 2nd Edition. Institut Français d’Afrique Noir, Dakar, 190 pp. Wallach, V., Wüster, W. & Broadley, D.G. (2009) In praise of subgenera: taxonomic status of cobras of the genus Naja Laurenti (Serpentes: Elapidae). Zootaxa, 2236, 26–36. Wüster, W. & Broadley, D.G. (2003) A new species of spitting cobra (Naja) from north-eastern Africa (Serpentes: Elapidae). Journal of Zoology, 259, 345–359. https://doi.org/10.1017/S0952836902003333 Wüster, W. & Broadley, D.G. (2007) Get an eyeful of this: a new species of giant spitting cobra from eastern and north-eastern Africa (Squamata: Serpentes: Elapidae: Naja). Zootaxa, 1532, 51–68. Wüster, W., Crookes, S., Ineich, I., Mané, Y., Pook, C.E., Trape, J.-F. & Broadley, D.G. (2007) The phylogeny of cobras inferred from mitochondrial DNA sequences: Evolution of venom spitting and the phylogeoggraphy of the African spitting cobras (Serpentes: Elapidae: Naja nigricollis complex). Molecular Phylogenetics and Evolution, 45, 437–453. https://doi.org/10.1016/j.ympev.2007.07.021 Zheng, H. & Zhang, Q. (2000) Medicinal using status and conservation strategy of snakes in China. In: Zhang, E. & Zheng, H. (Eds.), Conservation of endangered wild medicinal fauna and Xora resources in China. Second Military Medical University Press, Shanghai, pp. 96–101. Zhou, Z. & Jiang, Z. (2005) Identifying snake species threatened by economic exploitation and international trade in China. Biodiversity and Conservation, 14, 3525–3536. https://doi.org/10.1007/s10531-004-0826-5

APPENDIX 1. List of the examined specimens.

Naja peroescobari sp. nov. São Tomé Island: Museu Nacional de História Natural e da Ciência: MUNHAC/MB03-001065 [Praia Inhame, N: 0.028636, E: 6.523203, WGS-84] (holotype); MUNHAC/MB03-000948, MUNHAC/MB03-000949, MUHNAC/MB03- 000950 [Jardim Botânico, N: 0.27427, E: 6.58581, WGS-84]; MUNHAC/MB03-000947 [trail to lagoon Amélia; N: 0.26800, E: 6.59131, WGS-84]; MUNHAC/MB03-000985 [São João dos Angolares, N: 0.133333, E: 6.650000. Instituto de Investigação Ciência Tropical: IICT 50-1954 [Nova Moka; N: 0.28736, E: 6.3342, WGS-84]; IICT 2-1966 [without specific locality] (paratype); IICT 20-1967 [Santa Josefina, N: 0.247882, E: 6.738315, WGS-84] (paratype); IICT 18-1972 [Ribeira

140 · Zootaxa 4324 (1) © 2017 Magnolia Press CERÍACO ET AL. Peixe, N: 0.090278, E: 6.615278, WGS-84] (paratype). Muséum d´histoire naturelle de la ville de Genève: MHNG 2518.41, MHNG 2518.42 [Rio Angra Toldo, N: 0.156881, E: 6.668792 WGS-84] (paratype); MHNG 2462.43 [environs of Neves, N: 0.353192, E: 6.634972 WGS-84] (paratype). Zoological Museum of the University of Hambourg: ZMH R10526 [without specific locality] (paratype); ZMH R10527, ZMH R10528 [without specific locality].

Naja melanoleuca Hallowell, 1857 Angola: Zoologische Staatssammlung München: ZSM 101/1954 [Piri Dembos, S: 8.53333, E: 14.43333 WGS-84]. Zoological Museum of the University of Hamburg: ZMH 281a, ZMH 281b [Libolo/Luati, S: 9.98333, E: 14.90000]; ZMH 186 [Piri Dembos, S: 8.53333, E: 14.43333 WGS-84]. : Zoologische Staatssammlung München: ZSM 6/1984 [NE Bujumbura, N: 3.3822, E: 29.3644]. Cameroon: American Museum of Natural History: AMNH 51815, AMNH 51816, AMNH 51817, AMNH 51818, AMNH 51819, AMNH 51820 [Metet, N: 7.083333, E: 13.283333 WGS-84]. Museum für Naturkunde, Berlin: ZMB 20885, ZMB 22886 [without specific locality]. Zoologische Staatssammlung München: ZSM 10/1978, ZSM 2215/0, ZSM 2216/0, ZSM 2219/0 [Undetermined locality]. Museum of Comparative Zoology: MCZ 10071 [Lolodrof, N: 3.233333, E: 10.733333 WGS- 84]; MCZ 13216 [Metet, N: 7.083333, E: 13.283333 WGS-84]. Central African Republic: American Museum of Natural History: AMNH 120491, [Bambari, N: 5.767948, E: 20.675651 WGS-84]. Democratic Republic of Congo: Academy of Natural Sciences of Philadelphia: ANSP 20746 [Saidi’s Village]. American Museum of Natural History: AMNH 12319, AMNH 13220, AMNH 11321, AMNH 12322, AMNH 12324, AMNH 12368, AMNH 12369, AMNH 12370, AMNH 12373, AMNH 12374, AMNH 12376, AMNH 12377, AMNH 12378, AMNH 12380, AMNH 12381 [without specific locality]; AMNH 12372 [Medje, N: 2.416667, E: 27.3 WGS-84]; AMNH 12374 [Niapu, N: 2.416667, E: 26.466667 WGS-84]. Museum of Comparative Zoology: MCZ 24746 [Bumba, N: 2.183333, E: 22.466667 WGS- 84]; MCZ 25958 [Buta, near Uele Territory N: 2.8, E: 24.733333 WGS-84]. Zoologische Staatssammlung München: ZSM 293/ 1912 [Rulei, Uille district]. : American Museum of Natural History: AMNH 16935, AMNH 16936 [Benito, N: 1.50000, E: 9.750000 WGS-84]. Gabon: Academy of Natural Sciences of Philadelphia: ANSP 6875, ANSP 6876, ANSP 6878, ANSP 6879 [without specific locality] (syntypes of Naja haje var. melanoleuca). Ghana: Academy of Natural Sciences of Philadelphia: ANSP 27251 [Ghana?]. Museum of Comparative Zoology: MCZ 49083 [Tafo, N: 6.22379, E: 0.366568 WGS-84]. Museum of Vertebrate Zoology: MVZ 249816 [Ghana, Volta Region, N: 8.2595, E: 0.52203]; MVZ245386 [Ghana, Volta Region, N: 8.26515, E: 0.51753]. Liberia: American Museum of Natural History: AMNH 104366 [Grand Gedeh N: 5.916667, E: 8.083333 WGS-84]. Togo: Museum für Naturkunde, Berlin: ZMB 28485 [without specific locality]. Uganda: Academy of Natural Sciences of Philadelphia: ANSP 20781 [West Mengo, Kitala district, N: 0.116667, E: 32.516667 WGS-84].

Naja subfulva Laurent, 1955 Angola: Zoologische Staatssammlung München: ZSM 100/1954 [Piri Dembos, S: 8.53333, E: 14.43333 WGS-84]. Zoological Museum of the University of Hamburg: ZMH 81, ZMH 98, ZMH 184 [Piri Dembos, S: 8.53333, E: 14.43333 WGS- 84]; ZMH 280 [Libolo/Luati, S: 9.98333, E: 14.90000 WGS-84]. Museum für Naturkunde, Berlin:ZMB 46561 [Cacuso, S: 9.421023, E: 15.746679 WGS-84]. Cameroon: Museum für Naturkunde, Berlin: ZMB 18524 [Kribi, N: 2.940488, E: 9.909771 WGS-84]. Liberia: Museum of Comparative Zoology: MCZ 22532 [North of Moyla, N: 6.728889, E: 10.644722 WGS-84]; MCZ 51634 [Gbangam 3 miles NE of Suacoco, N: 7.00646, E: 9.483208 WGS-84]. Mozambique: Museum of Vertebrate Zoology: MVZ266146 [Zambezia province, N: -15.438946, E: 36.89756] Tanzania: Zoologische Staatssammlung München: ZSM 95/1936 [Gomberi].

COBRA-PRETA OF SÃO TOMÉ ISLAND Zootaxa 4324 (1) © 2017 Magnolia Press · 141