Endangered beauties: micro-CT cranial osteology, molecular genetics and external morphology reveal three new species of chameleons in the Calumma boettgeri complex (Squamata: Chamaeleonidae) Author David Prötzel Author Miguel Vences Author Oliver Hawlitschek Author Mark D. Scherz Author Fanomezana M. Ratsoavina Author Frank Glaw text Zoological Journal of the Linnean Society 2018 2018-04-09 2018 20 1 28 journal article 30217 10.1093/zoolinnean/zlx112 99b3b009-f006-4146-b335-cbfdc2fcd60b 1226487 0786ACDD-7205-4125-B495-A74601AF1815 CALUMMA UETZI SP. NOV. urn:lsid:zoobank.org:act:C13858A2-8989-475E-9679- F96F283E34D7 Holotype : ZSM 1688 / 2012 ( FGZC 3627 ), adult male with completely everted hemipenes (hemipenis on the right removed for micro-CT scanning), collected in the Sorata massif ( 13.6944°S , 49.4441°E , 1100 m a.s.l.), Antsiranana Province , northern Madagascar , on 27 November 2012 by F. Glaw , O. Hawlitschek , T. Rajoafiarison , A. Rakotoarison , F. M . Ratsoavina , and A. Razafimanantsoa . Paratypes : ZSM 1686 / 2012 ( FGZC 3674), subadult male, collected in Sorata above the campsite (13.6804° S , 49.4395° E , 1396 m a.s.l.) on 28 November 2012 ; ZSM 1685 / 2012 ( FGZC 3593), adult female, collected in Sorata above the campsite (13.6805° S , 49.4451° E , 1417 m a.s.l.) on 26 November 2012 ; ZSM 1687 / 2012 ( FGZC 3640), adult female, collected in Sorata, bamboo forest above the campsite (13.6746° S , 49.4406° E , 1516 m a.s.l.) on 28 November 2012 ; UADBA-R uncatalogued (FGZC 3628), adult female, collected in Sorata (13,6944° S , 49,4441° E , 1100 m a.s.l.) on 27 November 2012 ; all collected by F . Glaw , O . Hawlitschek, T . Rajoafiarison, A . Rakotoarison, F . M . Ratsoavina, and A . Razafimanantsoa. Referred material: ZSM 450/ 2016 ( ZCMV 15287), juvenile, collected in Marojejy National Park at Camp 3 ‘Simpona’ ( 14.43661° S , 49.74335° E , 1325 m a.s.l.) on 18 November 2016 by M . D. Scherz, A. Rakotoarison, M . Bletz, M . Vences, A . Razafimanantsoa, and J . Razafindraibe. This juvenile specimen was recently identified by DNA barcoding to be conspecific with Calumma uetzi but is not figured above. Diagnosis: Calumma uetzi sp. nov. is a member of the phenetic C. nasutum species group ( Prötzel, Ruthensteiner & Glaw, 2016 ), on the basis of the presence of a soft, dermal unpaired rostral appendage, absence of gular or ventral crests, and heterogeneous scalation on the lower arm, consisting mostly of tubercles of diameter 0.3–0.5 mm . Within the group, it is one of the smallest species, at 42.0– 45.7 mm SVL and 87.3–101.2 mm total length. The brown to olive-green chameleon is characterized by a long and distally rounded rostral appendage, occipital lobes that are clearly notched but not completely separated, a distinctly elevated rostral crest, a dorsal crest in both sexes, absence of axillary pits and unique skull morphology. Table 3. Mean interspecific genetic distances, and mean and maximal intraspecific genetic distances between species of the Calumma boettgeri complex (uncorrected pairwise distances of a fragment of the mitochondrial ND2 gene, expressed as a percentage)

C. boettgeri	C. gehringi	C. guibei	C. juliae	C. lefona	C. linotum	C. uetzi
C. boettgeri	2.1/2.1
C. gehringi	17.1	6.6/11.4
C. guibei	19.0	14.8	4.1/6.8
C. juliae	11.2	12.6	15.4	0.0/0.0
C. lefona	17.4	14.2	9.2	14.5	NA
C. linotum	12.1	15.4	17.7	10.9	15.1	4.6/10.1
C. uetzi	15.3	12.2	14.9	12.2	13.5	12.4	0.0/0.0

Calumma uetzi sp. nov. differs from C.fallax , C.gallus , C. nasutum , Calumma peyrierasi (Brygoo, Blanc & Domergue, 1974) , C. vatosoa, and C. vohibola of the C. nasutum group by the presence of occipital lobes; from C. linotum and C. boettgeri in the clearly notched occipital lobes with a depth of 0.5–1.0 mm (vs. not or slightly notched with 0–0.3 mm and 0–0.6 mm ); additionally, from C. boettgeri by the higher number of large juxtaposed tubercle scales on the extremities ( 15–18 in line vs. 7–14, isolated from each other). From the most similar taxa, C. gehringi and C. guibei ( Prötzel et al. , 2017 ), C. uetzi sp. nov. differs in the smaller body size of 42.0– 45.7 mm SVL in adults (vs. 47.5–53.7 mm in C. gehringi and 48.1–53.7 mm in C. guibei ); a longer tail relative to SVL in females of 108–118% (vs. 89–106% in C. gehringi and 95–101% in C. guibei ); the presence of a dorsal crest in females (vs. absence); a straight-lined dorsal margin of the supralabial scales vs. serrated (character ‘en dents de scie’ of Angel, 1942); furthermore, in skull morphology of adult males of C. gehringi and C. guibei ( Prötzel et al. , 2017 ), by a completely closed brain case in adults (vs. presence of a frontoparietal fenestra with a diameter of 0.7– 2.6 mm ); elevated protuberances at the anterior end of the maxillae in males (vs. absence); and in a broadened parietal with a width at its midpoint of 24.8% related to skull length (vs. 9.9–15.3%). Furthermore, C. uetzi sp. nov. differs from all other species by a unique coloration of males in life. Description of the holotype ( Figs 2 , 3A ) : Adult male, in a good state of preservation; mouth slightly opened, with tongue tip between the jaws; originally, both hemipenes completely everted, but right hemipenis removed for micro-CT scanning and stored in a separate Eppendorf tube alongside the specimen; SVL 45.7 mm , tail length 55.5 mm ; for other measurements, see Table 1 ; distinct and elevated rostral ridges that form a concave cup on the snout and fuse on the anterior snout at the base of a tapering, laterally compressed dermal rostral appendage that projects straight forward over a length of 3.8 mm beyond the snout tip with a diameter of 2.0 mm, rounded distally; 12 infralabial and 11 supralabial scales; supralabials with a straight dorsal margin; no supra-orbital crest; distinct lateral crest running horizontally; short temporal crest consisting of two tubercles per side; indistinct parietal crest; occipital lobes clearly developed and separated, but still slightly connected, by a deep, V-shaped notch (1.0 mm); casque raised; dorsal crest present, starting 0.9 mm from the base of the notch between the occipital lobes, consisting of a row of 13 separated, small conical scales spaced at irregular intervals from 0.1 to 1.8 mm ; no caudal crest; no traces of gular or ventral crest. Body laterally compressed, with fine homogeneous scalation with the exception of the extremities and head region; limbs with rounded tubercle scales having a maximal diameter of 0.5 mm ; heterogeneous scalation on the head, with largest scale on temporal region with diameter of 1.0 mm; 28 oval tubercle scales (diameter> 0.3 mm ) per side on rostral appendage; no axillary or inguinal pits. Skull osteology of the holotype ( Fig. 4A ; Supporting information, Video S1) : Skull length 10.9 mm ; snout– casque length 12.9 mm ; maxillae with elevated protuberances at the anterior end; narrow paired nasals separated from each other; anterior tip of frontal exceeding more than half of the naris and meeting the premaxilla; prefrontal fontanelle and naris separated by contact of prefrontal with maxilla; prominent prefrontal with laterally raised tubercles; frontal and parietal with several tubercles; frontal with a width of 2.8 mm (25.7% of skull length) at border to prefrontal, extending to 4.3 mm (39.4%) at border to postorbitofrontal; broad parietal, tapering more or less constantly from a width of 4.2 mm (38.5%) at the border to frontal and still broad at midpoint with 2.7 mm (24.8%) until it meets the squamosals, then narrowing rapidly to a tip; posterodorsally directed parietal meets the squamosal laterally; squamosal thick, with several tubercles. For further measurements, see Table 2 . Coloration of the holotype in preservative ( Fig. 3A ) : The body of the holotype in preservative is of grey–blue colour, with an indistinct beige lateral stripe and a light blueish stripe above it; ventral and temporal region and gular folds also beige; extremities speckled with blue to light blue tubercle scales; tail irregularly annulated beige and grey. Variation : The three ZSM paratypes agree well with the holotype in most characters of morphology and osteology: male ZSM 1686/2012 with a dorsal crest of elongated spines ( Fig. 3A ; vs. conical scales in holotype ) and a small number of tubercle scales on the rostral appendage (19 per side); temporal crest varies between the specimens from one to two tubercles; depth of notch of occipital lobes is smaller in paratypes ( 0.2–0.6 mm ; juvenile specimens included); number of dorsal cones in males is distinctly higher (13–14) than in females (three to five; juvenile ZSM 1685/2012 assumed as female). The skull of a second micro-CT-scanned specimen (ZSM 1686/2012) differs by the smooth frontal and parietal that is also narrower at its midpoint (3.2% of SVL, vs. 4.4% of SVL in the holotype ). Both characters can be attributed to the subadult developmental stage of the specimen. Coloration in life (based on observations and photographs of the type specimens only; Fig. 2 ) : Sexes are dichromatic, with males in relaxed state generally greenish beige and females with brown body coloration; a beige–white lateral stripe may occur from the occipital lobes to the hip. In males, the stripe is distinct and framed by a violet line. Throat and ventral region white in males, beige in females; rostral appendage not contrasted and of same colour as the body; dark lateral stripe from nostrils, crossing the eyes to the base of the occipital lobes; cheek region highlighted in blue (males) or green (females) colour; large scales on temporal region of males of bluish colour. Signalling males show a spectacular coloration of bright yellow all over the body, tail, extremities and eyelids, a red lateral stripe that is framed in violet, light blue cheek region, violet temporal region and a bluish–green rostral appendage; the eye-stripe darkens and contrasts the remaining head coloration. The stress coloration of females focuses only on the head region, with distinct yellow spots on the rostral appendage, rostral crest, dorsal head side and temporal crest; eyelids with radially aligned blue spots, also on occipital lobes; colourful spots contrasted by dark brown head coloration; rest of body uniformly brown ( Fig. 2C ). Hemipenial morphology based on diceCT scans ( Fig. 5A ; Supporting information, Video S2) : The hemipenis of Calumma uetzi sp. nov. shows large and deep calyces, with smooth ridges on the asulcal side of the truncus. The apex is ornamented with two pairs of pointed cornucula (recently described hemipenial ornament; see Prötzel et al. (2017) and paired rotulae. The cornucula rise from the sulcal side of the apex, are curved to the asulcal side and are not completely everted in the investigated specimen. A pair of rotulae, with four and six tips, respectively, is placed on the asulcal side. Additionally, on the asulcal side there is a pair of larger rotulae that are curved and bear 12 and 13 tips. The top of the apex has a papillary field of several fleshy papillae. Available names : Apart from C. gehringi and C. guibei , there is no other valid species or synonym in the C. nasutum group with deeply notched occipital lobes. Figure 2. Calumma uetzi sp. nov. in life. A, male (ZSM 1686/2012) in slightly stressed coloration. B, subadult female (ZSM 1685/2012) relaxed. C, male holotype (ZSM 1688/2012, left) in spectacular display, with adult female (right, UADBA- R-FGZC 3628) in stress coloration, repelling the male. Etymology : This species is dedicated to our colleague and friend Peter Uetz, who developed and has maintained the Reptile Database (http://www. reptile-database.org/) voluntarily for> 20 years. This database is the most important online resource for information on reptile species, thereby providing a priceless service to herpetology and a model for what should be available for all organism groups. Distribution : Calumma uetzi sp. nov. was originally discovered in northern Madagascar , in the area of the Sorata massif ( Antsiranana Province ) from elevations of 1100–1500 m a.s.l., but new DNA barcoding results revealed that a juvenile chameleon recently collected in Marojejy National Park ( 14.43661°S , 49.74335°E , 1325 m a.s.l.; Fig. 6 ) also belongs to C. uetzi sp. nov. Natural history and ecology : The principal habitat of Calumma uetzi sp. nov. is primary mid-elevation rainforest that has recently been degraded at different levels. Most individuals were found at night roosting in the vegetation on thin branches or on the tip of leaves ~ 1–3 m above the forest floor and had red mites between the fingers and toes. When a male and a female were put in close proximity on the same branch, both sexes quickly changed colour and became brightly coloured ( Fig. 2 ), and the female threatened the male with an open mouth. In other cases, one of the individuals moved away to avoid closer contact. In one, case the artificial encounter led to a possible mating attempt. Recommended IUCN status : Given the limited data available on C. uetzi sp. nov. , specifically no data pertaining to the size of its population or probability of extinction, it cannot currently be assessed under any criterion other than B of the IUCN Red List Criteria ( IUCN, 2012 ). A minimal convex polygon of the Sorata and Marojejy massifs, corresponding to the estimated extent of occurrence (EOO) of the species (criterion B1), covers an area of ~ 2500 km 2. It is currently known from two threat-defined locations (as defined by the IUCN, 2012 ; criterion B, subcriterion a), one of which is well protected (Marojejy National Park). The other location (Sorata) is not yet protected, but is currently included in a planned protected area, COMATSA Nord (corridor between Marojejy, Anjanaharibe-Sud and Tsaratanana protected areas, WWF Madagascar , 2015 ). At present, Sorata is experiencing high deforestation pressure, and forest has been largely eradicated from sea level to ~ 1200 m a.s.l. As such, the deforestation pressure is directly impacting the distribution range of C. uetzi sp. nov. in this area [criterion B, subcriterion b(iii)]. As the EOO of the species is < 5000 km 2, and the number of threatdefined locations is fewer than five, the species qualifies as Endangered under IUCN criterion B1ab(iii).