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 LEFONA SP. NOV. urn:lsid:zoobank.org:act:99AF4F23-EBDF-4276-AE41- 3C04E7443FD8 Remark: This species was considered as clade FII by Gehring et al. ( 2012 ). Holotype : ZSM 2 8 4 9/ 2 0 1 0 (DRV 6 2 8 7), adult male with incompletely everted hemipenes, left hemipenis removed for micro-CT scanning, collected in Andrevorevo southeast of Tsaratanana Massif ( 14.3464° S , 49.1028° E , 1717 m a.s.l. ), Mahajanga Province , northern Madagascar , on 21 June 2010 by F . M . Ratsoavina and F . Randrianasola. Paratypes : None. Diagnosis : Calumma lefona 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 ~ 0.5 mm diameter. Within the genus, it is a small sized chameleon (SVL 51.3 mm , TL 113.7 mm ) that is characterized by a long and pointed rostral appendage, occipital lobes that are widely notched and completely separated, a distinctly elevated rostral crest, a dorsal and caudal crest, absence of axillary pits and unique skull morphology. Calumma lefona sp. nov. differs from C. fallax , C. gallus , C. nasutum , C. peyrierasi , C. vatosoa, and C. vohibola of the C. nasutum group by the presence of occipital lobes; from C. boettgeri and C. linotum in the clearly notched occipital lobes with a depth of 1.8 mm (vs. not or slightly notched with 0–0.7 mm ), presence of a frontoparietal fenestra with a width of 1.9 mm (14.7% of skull length; vs. completely closed brain case), prefrontal, frontal and parietal with many tubercles (vs. smooth or only a few tubercles); additionally, from C. boettgeri by the higher number ( 21 in line) of large, juxtaposed tubercle scales on the extremities (vs. seven to 14 isolated tubercles). From the other three taxa with notched occipital lobes, C. gehringi , C. guibei, and C. uetzi sp. nov. , C. lefona sp. nov. differs by the long ( 5.6 mm ) and pointed rostral appendage (vs. 3.1–5.4 mm in males, rounded), with 60 large scales (diameter> 0.3 mm ) on the right side of the rostral appendage (vs. 20–42 in males), dorsal crest of 23 small conical scales (vs. absence or 5–15 large conical scales), clearly and widely separated occipital lobes with a notch of 1.8 mm (vs. tightly separated or connected with a notch of 0.5–1.5 mm in males); furthermore, in skull morphology by a narrower frontal, e.g. at the border to the postorbitofrontal with 30.5% of SkL (vs. 35.5–39.7%) and strongly raised maxillae; from C. gehringi and C. guibei in a broader parietal at midpoint with 18.3% of SkL (vs. 9.9–15.3%); from C. uetzi sp. nov. by possession of a frontoparietal fenestra (vs. completely closed brain case) and a narrower head (e.g. RFWPo of 30.5 vs. 35.6–39.4%); from C. guibei by a smaller frontoparietal fenestra with 14.7% of SkL (vs. 21.0%), prefrontal fontanelle and naris separated by contact of prefrontal with maxilla (vs. fused), thick squamosal (vs. thin) in broad dorsal contact with the parietal (vs. not meeting parietal or only in weak contact); and from C. gehringi by possession of a larger frontoparietal fenestra (21.0% of SkL vs. 5.8–9.1%). Description of the holotype ( Fig. 3B ) : Adult male, in a good state of preservation, left forelimb removed for DNA analysis, left hemipenis removed for micro-CT scanning; mouth slightly opened with tongue between the jaws; originally both hemipenes incompletely everted ( Fig. 5B ), but left hemipenis cut off for micro-CT scanning and stored in a separate Eppendorf tube alongside the specimen; SVL 51.3 mm , tail length 62.4 mm ; see Table 1 for other measurements; 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 5.6 mm with a diameter of 2.0 mm, pointed distally; 12 infralabial and 11 supralabial scales; supralabials dorsally serrated; 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 completely separated by a deep, ‘U’-shaped notch ( 1.8 mm ); casque raised; dorsal crest present, starting 0.8 mm from the base of the notch between the occipital lobes, consisting of a row of 23 separated, small conical scales spaced at irregular intervals from 0.1 to 0.9 mm , and several more on the tail decreasing in size towards the tip; 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 with a maximal diameter of 0.5 mm ; heterogeneous scalation on the head, with largest scale on temporal region with diameter of 0.6 mm and 60 oval tubercle scales (diameter> 0.3 mm ) on the right side of the rostral appendage; no axillary or inguinal pits. Figure 3. Holotypes of the three new Calumma species as preserved specimens. A, C. uetzi sp. nov. (ZSM 1688/2012). B, C. lefona sp. nov. (ZSM 2849/2010). C, C. juliae sp. nov. (ZSM 143/2016). Scale bar: 20 mm. Figure 4. Micro-computed tomography scans of the skulls of the three holotypes in dorsal and lateral view. A, male Calumma uetzi sp. nov. (ZSM 1688/2012). B, male C. lefona sp. nov. (ZSM 2849/2010). C, female C. juliae sp. nov. (ZSM 143/2016). D, male C. boettgeri (ZSM 440/2000). E, male C. linotum holotype. F, female C. linotum (ZSM 551/2001). G, male C. gehringi holotype (ZSM 2851/2010). H, male C. guibei (ZSM 2855/2010). Diagnostic characters are encircled in red. Abbreviations are given in the Material and Methods. Scale bars: 2.0 mm. Skull osteology of the holotype ( Fig. 4B , Supporting information, Video S3) : Skull length 13.1 mm ; snout–casque length 15.9 mm ; broad paired nasals meeting anteriorly, anterior tip of frontal exceeding more than half of the naris; prefrontal fontanelle and naris separated by contact of prefrontal with maxilla; elevated maxillae building a rectangular edge at anterior margin; prominent prefrontals with laterally raised tubercles; frontal and parietal with several tubercles, five on the midline forming a parietal crest; frontal with a width of 2.9 mm (22.1% of SkL) at border to prefrontal extending to 4.0 mm (30.5% of SkL) at border to postorbitofrontal; frontoparietal fenestra with transverse diameter of 1.9 mm (14.7% of SkL); parietal tapering more or less constantly, with a width of 3.9 mm (29.8% of SkL) at the border to frontal and 2.4 mm (18.3% of SkL) at its midpoint, and then tapering rather strongly to the posterior 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. 3B ) : The body of the holotype in preservative is of a grey–blue colour, with an indistinct beige lateral stripe; ventral and temporal regions and throat also beige; dark blue line from dorsal part of rostral appendage crossing the eyes and the lateral crest, ending in occipital lobes; extremities speckled with bluish tubercle scales. The coloration in life is unknown. Hemipenial morphology based on diceCT scans : The hemipenis of the holotype was incompletely everted, and a diceCT scan resulted in an inadequate illustration ( Fig. 5B ). Apparently, it showed deep calyces and two pairs of rotulae; changing the threshold revealed two pairs of cornucula that were not everted. Available names : Apart from C. gehringi , C. guibei, and C. uetzi sp. nov. , there is no other valid species or synonym in the C. nasutum group with deeply notched occipital lobes. Etymology : Calumma lefona sp. nov. is the only species in the C. nasutum group with a relatively long and pointed/constantly tapering rostral appendage (with the exception of C. gallus ). This shape reminds of a spearhead; accordingly, we chose the Malagasy word ‘lefona’ (meaning ‘spear’) as its species epithet. It is used as an invariable noun in apposition to the genus name. Distribution : Calumma lefona sp. nov. is, so far, known from only a single location in northern Madagascar , southeast of the Tsaratanana Massif, called Andrevorevo, at 1717 m a.s.l. ( Fig. 6 ; for geographical coordinates see the ‘holotype’ details above). It lives within the lower elevational range of its sister taxon C. guibei (from 1590–2250 m a.s.l.) and at higher elevation than C. gehringi ( 730–1540 m a.s.l.). Natural history and ecology : Calumma lefona sp. nov. was found roosting at night on tree branches ~ 2 m above the ground. The habitat consisted of primary forest with closed canopy cover and small streams. During the nocturnal observations on 20 and 21 June 2010 , two adults and two juveniles were found along the forest path, but only one adult (the holotype ) was collected. Recommended IUCN status : Practically no data are available on the distribution and condition of C. lefona sp. nov. , and the species is known from a single specimen and a few additional observations. To avoid inflation of perceived risk, we recommend that this species be considered Data Deficient by the IUCN until more data become available.