Wednesday, August 1, 2018

[Herpetology • 2018] Integration of Nuclear and Mitochondrial Gene Sequences and Morphology Reveals Unexpected Diversity in the Forest Cobra (Naja melanoleuca) Species Complex (Serpentes: Elapidae) in Central and West Africa: Naja (Boulengerina) guineensis & N. (B.) savannula


[upper left] Naja (Boulengerina) guineensis Broadley, Trape, Chirio, Ineich & Wüster, 2018
[upper right] Naja (Boulengerinasavannula Broadley, Trape, Chirio & Wüster, 2018

[lower] Naja (Boulengerinamelanoleuca Hallowell, 1857

in Wüster, Chirio, Trape, Ineich, Jackson, et al., 2018. 

Abstract
Cobras are among the most widely known venomous snakes, and yet their taxonomy remains incompletely understood, particularly in Africa. Here, we use a combination of mitochondrial and nuclear gene sequences and morphological data to diagnose species limits within the African forest cobra, Naja (Boulengerinamelanoleuca. Mitochondrial DNA sequences reveal deep divergences within this taxon. Congruent patterns of variation in mtDNA, nuclear genes and morphology support the recognition of five separate species, confirming the species status of N. subfulva and N. peroescobari, and revealing two previously unnamed West African species, which are described as new: Naja (Boulengerinaguineensis sp. nov. Broadley, Trape, Chirio, Ineich & Wüster, from the Upper Guinea forest of West Africa, and Naja (Boulengerinasavannula sp. nov. Broadley, Trape, Chirio & Wüster, a banded form from the savanna-forest mosaic of the Guinea and Sudanian savannas of West Africa. The discovery of cryptic diversity in this iconic group highlights our limited understanding of tropical African biodiversity, hindering our ability to conserve it effectively.

Keywords: Integrative taxonomy, Africa, Naja melanoleucaNaja guineensis sp. nov., Naja savannula sp. nov., Elapidae, systematics, Reptilia

FIGURE 5. Naja (Boulengerina) guineensis sp. nov. Top: holotype, MNHN 1921.0485, dorsal and ventral view and side view of head. Note extensive mottling of throat and anterior ventral side and limited posterior extent of lighter ventral markings.
Bottom: live adult specimen measuring approximately 200 cm total length, from Sekondi-Takoradi, Western Region, Ghana, displaying dark suffusion of throat and anterior venter (not preserved; photo L. Chirio).

Naja (Boulengerina) guineensis sp. nov. 
Broadley, Trape, Chirio, Ineich & Wüster 
Naia melanoleuca (not Hallowell) Boulenger, 1896: 376 (part, var. B [c,d], C). 
Naja sp. 2 cf. melanoleuca Hallowell, 1857 (blackish dorsum) Trape & Baldé, 2014: 318. 
Naja sp. 2 cf. melanoleuca (forest form). Trape & Baldé, 2014: 336.

Diagnosis. Naja guineensis can be distinguished from the partly sympatric N. savannula sp. nov. by lacking extended dorsal banding, often having 17 rather than 19 dorsal scale rows at midbody, a generally lower subcaudal scale count, fewer ventral bands, a lesser posterior extent of the ventral banding, and a strong tendency towards melanism in adults. Specimens with 19 midbody dorsal scale rows can be distinguished from N. melanoleuca through the reduced number of ventral bands, lesser posterior extent of banding and tendency of ontogenetic melanism from N. subfulva in lacking a lighter anterior dorsum and through ontogenetic melanism, and from N. peroescobari in having the posterior chin shields in contact. 

Variation. Midbody dorsal scale row counts of 17 and 19 are approximately equally common in this species. In large adults, light pattern elements on the head and throat often become heavily suffused with black pigment, leading to a virtually entirely melanistic snake. Some specimens have 1–4 generally faint or poorly defined light bands across the neck, and occasionally an ocellate hood marking. 

Largest recorded: 1818+437 = 2255 mm, from Ballassou, Guinea (IRD 4213.G), but larger specimens have been observed. Based on locality, Menzies’ (1966) report of a specimen measuring “eight feet, eight inches” (264 cm) from Bo, Sierra Leone, is likely to refer to this species. 

Etymology. The specific epithet guineensis means “from Guinea” and is chosen to reflect the distribution of the species in the Upper Guinea forests of West Africa, part of the West African Forests biodiversity hotspot (Myers et al., 2000).
 Suggested common name. Black forest cobra. 

Distribution. The distribution of Naja guineensis appears to be restricted to the Upper Guinea Forests of western Africa, from western Togo to Liberia and Guinea (Trape & Baldé, 2014) (Fig. 6). There is a single record from Contuboel, Guinea Bissau (MBL 535). All other records 10°N or lower. 


FIGURE 7. Naja (Boulengerina) savannula  sp. nov.
 Top: holotype, MNHN 2018.0002. Bottom: live specimen from Kindia, Guinea, showing conspicuous, broad dorsal bands and ventral banding, including narrow accessory bands (not vouchered). Photos J.-F. Trape.

Naja (Boulengerina) savannula sp. nov. 
Broadley, Trape, Chirio & Wüster 
Naia melanoleuca (not Hallowell) Boulenger, 1896: 376 (part, var. D). 
Naja “banded form” Hughes, 2013: 128. 
Naja sp. 1 cf. melanoleuca Hallowell, 1857 (yellow banded dorsum) Trape & Baldé, 2014:318.
 Naja sp. 1 cf. melanoleuca (banded savanna form) Trape & Baldé, 2014: 336.  

Diagnosis: Distinguishable from other species of the N. melanoleuca complex by the presence of 3 to 8 semidivided yellowish or whitish bands on the anterior dorsal forebody, becoming uniform black caudad; venter yellow with 2 to 8 black bands. Generally higher mean subcaudal scale counts than the other species. Genetically diagnosable through possession of unique mitochondrial haplotypes (cytochrome b: GenBank MH337597–602; ND4: MH337403–408) and unique PRLR and UBN1 haplotypes (PRLR: MH337501–504; UBN1: MH337532– 535).

Variation: Dorsal scale rows on neck 19–25, at midbody 19, before vent 12–15; ventrals 211–233, subcaudals 63–77 (Table 8). Dorsal semi-divided yellow bands 3–8; ventral principal black bands 2–8 (Fig. 7).

 Largest recorded: 1825+405 = 2230 mm, from Medina Djikoye, Senegal (IRD 6155.S). 

Etymology: The name is derived from the contraction of its savanna habitat and annulated colour pattern and was coined by Barry Hughes in an unpublished 1968 manuscript. We have retained this name at the request of our colleague Barry Hughes.

Suggested common name: West African banded cobra. 

Distribution: Senegal and Gambia east to northern Cameroon (Fig. 6). Naja savannula appears to be restricted to gallery forest areas in Africa in Guinean Forest/Savanna Mosaic, extending northwards into West Sudanian Savanna (Chirio, 2003, 2013; Monasterio et al., 2016). Our records are mostly from latitudes 10–14°N, except in the Dahomey Gap in eastern Ghana and Benin, where the species approaches the Gulf of Guinea Coast. The eastern extent of the range is poorly understood. A specimen from Margui Wandala district, northern Cameroon (approx. 10.5°N 13.6°E; MNHN 1962.0022) appears to be assignable to this species, and one of us (JFT) recently collected a specimen from Mboura, 20 km SW Baïbokoum, Logone Oriental Province, Chad (7.598°N, 15.596°E; IRD 2281.N), and there is a recent record from near Niamey, Niger (LC, unpublished data). It seems likely that the species has a wider distribution in northern Cameroon, extreme southern Chad and possibly even extreme northwestern Central African Republic (CAR).



FIGURE 6. Distribution of the five species of the Naja melanoleuca complex.

FIGURE 5. Naja (Boulengerina) guineensis sp. nov. Top: holotype, MNHN 1921.0485, dorsal and ventral view and side view of head. Note extensive mottling of throat and anterior ventral side and limited posterior extent of lighter ventral markings. Bottom: live adult specimen measuring approximately 200 cm total length, from Sekondi-Takoradi, Western Region, Ghana, displaying dark suffusion of throat and anterior venter (not preserved; photo L. Chirio).

FIGURE 7. Naja (Boulengerina) savannula  sp. nov.  Top: holotype, MNHN 2018.0002. Bottom: live specimen from Kindia, Guinea, showing conspicuous, broad dorsal bands and ventral banding, including narrow accessory bands (not vouchered). Photos J.-F. Trape.

FIGURE 8. Naja (Boulengerina) melanoleuca. Adult specimens from Yaoundé, Cameroon (left—photo J.-F. Trape) and Tsibilé, Gabon (right—photo L. Chirio). Note the diffuse but distinct hood mark that is often present in this species, and the combination of broad main bands and narrow accessory bands on the ventral side.

Naja (Boulengerina) melanoleuca Hallowell, 1857 

Naia haie var. melanoleuca Hallowell, 1857, Proc. Acad. Nat. Sci. Philadelphia: 61. 
Type locality: Gabon, syntypes ANSP 6875–76, 6878–79. 
Naja haje var. leucosticta Fischer, 1885, Jahr. Hamburg. Wiss. Anst. 2: 115, pl. v, fig. 11. Type locality: Cameroon and Ogooué River, Gabon, syntypes ZMH 4280, 7048, 7299–7302. 
Naia melanoleuca; Boulenger, 1896: 376 (part, vars. A [a,b] & B [a,b]) 
Naja melanoleuca melanoleuca; Laurent, 1956: 290, pl. xxvi, fig. 2. 
Naja (Boulengerinamelanoleuca; Wallach et al., 2009. 
Boulengerina melanoleuca; Wallach et al., 2014: 122. 
Aspidelaps bocagei Sauvage, 1884: 204 (type locality: Gabon and Majumba; holotype MNHN 1884.0015) has been listed as a synonym of N. melanoleuca (e.g., Broadley, 1983; Wallach et al., 2014; Ceríaco et al., 2017), but is in fact a synonym of Naja annulata, as is evident from Sauvage’s description, which notes approximately 20 dark double bands along the entire body length, and 21 mid-dorsal scale rows (see also Schmidt, 1923). 

Diagnosis: Dorsum black, often with 1–3 semidivided yellow crossbands on the neck, the first may be an ocellus; venter yellow with 4 to 6 black bands in the first 100 ventrals, thereafter uniform black. 

Variation: Dorsal scale rows on neck 19–27, at midbody 19 (very rarely 17 or 21); ventrals 209–230; subcaudals 59–74 (Table 8). Supralabials 7, the third and fourth entering the orbit; infralabials 8, the first four in contact with the anterior sublinguals, no cuneate; preocular 1; postoculars 3 (very rarely 2 or 4); temporals 1+2 or 1+3; nuchals bordering temporals 5–9, usually 7.

Colouration: Head brown, the supralabials barred black and yellow, chin yellow. Black above, sometimes a yellow monocellate marking on the hood, or 1–3 small yellow blotches, the dorsal scales may be tipped with white in juveniles (Fig. 8). Yellow or white below with 4 to 6 black bands on the first 100 ventrals, usually uniform black thereafter. 

Largest recorded: 2250+420 = 2670 mm, from Moniya, Ibadan, Nigeria (Butler, 1982: 110). 

Suggested common name: Central African forest cobra. 

Distribution: Centered on the Congo Basin, west to southwestern Nigeria and possibly southern Benin, south to northern Angola, not extending east of the Albertine Rift Valley, where it is replaced by N. subfulva. The western range limits are poorly understood. Populations from southeastern Nigeria are clearly assignable to this form. A few specimens from Lamta, southern Benin (IRD 12.B, IRD 54.B, IRD 60.B), and Ghana (MNHN 1983.0663–64; no further locality information) also appear to be assignable to N. melanoleuca. 


Naja (Boulengerina) subfulva Laurent, 1955

Suggested common name. Brown forest cobra. 

Diagnosis. Midbody scale rows 19, except along coastal regions of East Africa (Kenya, Tanzania), where most specimens have 17 rows. Pattern highly variable. Adults of most populations distinguishable in having a brown forebody, often with spots, generally becoming darker or blackish posteriorly. Labial pattern may be attenuated in many adults. Venter with several black, dark brown or greyish crossbands on the first 50 ventrals, gradually becoming uniform black caudad in some populations, but often remaining entirely light, often with extensive darker spotting or speckling. Where present, the light forebody and/or light posterior venter are diagnostic for this species. Generally fewer ventral bands and ventral scales than N. melanoleuca or N. savannula and fewer subcaudals than N. savannula (Table 8). Genetically diagnosable through possession of unique mitochondrial haplotypes (cyt b: GenBank MH337603–633; ND4: MH337409–439) and unique PRLR and UBN1 haplotypes (PRLR: MH337441–471; UBN1: MH337531, MH337536–562, MH337564–566). 

Variation. Dorsal scale rows on neck 19–27, at midbody 19 (very rarely 17 or 21); ventrals 196–226; anal entire; subcaudals 55–71 (Table 8). Supralabials 7 (very rarely 5 or 6), the third and fourth entering orbit; infralabials 8 (rarely 7), the first four (rarely three) in contact with the anterior sublinguals, no cuneate (very rarely one); preocular 1; postoculars 3; temporal 1+2 or 1+3; temporal bordering parietals 5–9, usually 7.


Naja (Boulengerina) peroescobari  
Ceríaco, Marques, Schmitz & Bauer, 2017

Photo: T. Pisoni. 
DOI: 10.11646/zootaxa.4324.1.7 

Naja (Boulengerina) peroescobari Ceríaco et al., 2017 

Diagnosis. In the original description, Naja peroescobari was diagnosed from the other members of the N. melanoleuca group through a lack of white ventrals posterior to ventral 22, a lack of lighter markings on the dorsum, and the separation of the posterior chin shields. Our sample does not fully support the diagnostic value of these characters: at least one specimen (BMNH 1906.3.30.80) has the posterior chin shields in contact, and discrete dark bands separated by lighter bands (although often suffused with dark brown pigment) extend as far back as ventrals 45 and 55, respectively, in BMNH 1906.3.30.80 and MBL 1954. Naja peroescobari is distinct from N. subfulva in never having a brown forebody or a light posterior venter and in lacking dark speckling or spotting on the forebody. It displays greatly reduced ventral banding compared to N. melanoleuca and N. savannula, and, unlike N. guineensis, never has 17 midbody dorsal scale rows. The species is also diagnosable through unique mitochondrial haplotypes (Ceríaco et al., 2017; cyt b: GenBank MH337634; ND4: MH337440) and a unique PRLR haplotype (MH337499) 

Variation. See Table 8 and Ceríaco et al. (2017) for variation in scale counts and ventral banding in this species.

 Distribution. Restricted to the volcanic island of São Tomé in the Gulf of Guinea, where it seems to inhabit primarily the forested parts of the centre and south of the island, whereas it seems to be missing from the more open northeast (Ceríaco et al., 2017).


Wolfgang Wüster, Laurent Chirio, Jean-François Trape, Ivan Ineich, Kate Jackson , Eli Greenbaum, Cesar Barron, Chifundera Kusamba, Zoltán T. Nagy, Richard Storey, Cara Hall, Catharine E. Wüster, Axel Barlow and Donald G. Broadley. 2018. Integration of Nuclear and Mitochondrial Gene Sequences and Morphology Reveals Unexpected Diversity in the Forest Cobra (Naja melanoleuca) Species Complex in Central and West Africa (Serpentes: Elapidae). Zootaxa. 4455(1); 68–98.  DOI: 10.11646/zootaxa.4455.1.3

Luis M. P. Ceríaco, Mariana P. Marques, Andreas Schmitz and Aaron M. Bauer. 2017. The “Cobra-preta” of São Tomé Island, Gulf of Guinea, is A New Species of Naja Laurenti, 1768 (Squamata: Elapidae). Zootaxa. 4324(1); 121–141.  DOI: 10.11646/zootaxa.4324.1.7

  

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