﻿Four new species of the genus Diploderma Hallowell, 1861 (Squamata, Agamidae) from China

﻿Abstract Four new species of Diploderma are described from Sichuan and Yunnan provinces, southwestern China, based on an integrative taxonomic approach, combining morphological and genetic data. The first new species from Danba County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.flaviceps, but it can be diagnosed from the latter by having a relatively much shorter tail and by a genetic distance of 4.4% in the ND2 gene; the second new species from Muli County, Sichuan Province, is phylogenetically closely related to D.daochengense, D.yongshengense, and D.yulongense, but it can be diagnosed from the latter three species by having a pale yellow gular spot and by genetic distances of 5.6–6.7% in the ND2 gene; the third new species from Jiulong County, Sichuan Province, is morphologically most similar and phylogenetically closely related to D.angustelinea, but it can be diagnosed from the latter by having a relatively much longer tail and by a genetic distance of 2.8% in the ND2 gene; and the last new species from Weixi County, Yunnan Province, is phylogenetically closely related to D.aorun, but it can be diagnosed from the latter by having a pale yellow gular spot and by a genetic distance of 2.9% in the ND2 gene. Our work brings the number of species within the genus Diploderma to 46.


Introduction
Currently the genus Diploderma Hallowell, 1861 comprises 42 recognized species (Cai et al. 2022;Liu et al. 2022;Wang et al. 2022b) which are mainly distributed in the hot-dry river valleys of the Hengduan Mountain Region of southwestern China, and more than half of them were described in the past decade (Wang et al. 2021a(Wang et al. , 2022aCai et al. 2022;Liu et al. 2022;Uetz et al. 2022).
The Hengduan Mountain Region contains many high mountains and rivers, forming complex hot-dry river valleys in this region, which are suitable for Diploderma species to inhabit. Therefore, it is not surprising that numerous new species of Diploderma were found in this region. However, due to the complex terrain and inconvenient transportation, there are still many areas of this region that have not been fully investigated for biodiversity.
During our field surveys in Yunnan and Sichuan provinces, China, in 2022, some specimens of Diploderma were collected from four different sites in the Hengduan Mountain Region. In terms of morphology, these specimens showed distinct differences to any recognized species of the genus. Furthermore, phylogenetic analyses indicated differentiation of these populations also at a molecular level. By comparing our data with morphological and molecular differentiation within existing species of Diploderma, we conclude that these populations have exceeded an intraspecific variation level and thus describe four new species of Diploderma herein.

Sampling
Field survey in Yunnan Province was conducted from June to July 2022 by Shuo Liu, and field survey in Sichuan Province was conducted from July to August 2022 by Mian Hou. Specimens were collected in the morning or afternoon from Weixi County, Yunnan Province, and Danba, Muli, and Jiulong counties, Sichuan Province, respectively (Fig. 1). Photographs were taken to document colour patterns in life prior to anaesthetisation and euthanasia, respectively. Genetic tissues were stored in 99% ethanol taken from livers, and specimens were preserved in 75% ethanol. All newly collected specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Morphological characteristics and analyses
Morphometric measurements and pholidosis data were taken for all newly collected specimens and 27 museum specimens deposited at KIZ. Specimens were measured using a digital caliper to the nearest 0.1 mm. Measurements were taken on the left side (unless the left side was damaged, then the right side was used), values for paired pholidosis characteristics were recorded on both sides and were provided in left/right order (following Liu et al. 2020Liu et al. , 2022Wang et al. 2022a, b). The following morphometric characteristics were measured and counted following Liu et al. (2022) and Wang et al. (2022a):

F4S
subdigital lamellae under fourth finger, subdigital lamellae scales from the base between third and fourth finger to the tip of fourth finger, excluding the claw; FLL fore-limb length, measured between the point of insertion at axillary to the tip of fourth finger, excluding the claw, measured on straightened limb; HD head depth, measured as the perpendicular distance at the temporal region of head; HL head length, measured from the tip of snout to the rear border of the angle of jaw; HLL hind limb length, measured between the point of insertion at groin to the tip of fourth toe, excluding the claw, measured on straightened limb; HW head width, measured between the widest points of the head;

IL
infralabial scale number, enlarged, modified labial scales from mental to the corner of mouth; MD middorsal crest scale number, modified crest scales longitudinally from the first nuchal crest to the scale above cloaca; NSL nasal-supralabials scale rows, number of horizontal rows of small scales between the first supralabial and the nasal; SEL snout-eye length, measured between the tip of snout and anterior edge of orbital bone; SL supralabial scale number, enlarged, modified labial scales from rostral to the corner of mouth; SOR suborbital scale rows, longitudinal rows of scales between supralabials and inferior-most edge of orbit circle, excluding fine ciliary scales in the orbit; SVL snout-vent length, measured from the snout tip to anterior edge of the cloaca; T4L fourth toe length, measured between the tip of fourth toe to the base between third and fourth toe, excluding the claw; T4S subdigital lamellae under fourth toe, subdigital lamellae scales from the base between third and fourth toe to the tip of fourth toe, excluding the claw; TAL tail length, measured from the anterior edge of the cloaca to the tip of tail; TRL trunk length, measured between the limb insertion points between axillary and groin; VN ventral scale number, ventral body scales counted in a straight line along the medial axis between the transverse gular fold and the anterior edge of cloaca.
Principal component analysis (PCA) was used to determine whether the newly collected specimens and species with closely morphological similarities to them occupied unique positions in morphospace, and whether the results coincide with the delineation of species as indicated by molecular phylogenetic analysis. Characters used in the PCA were mensural data from SVL, TAL, HL, HW, HD, SEL, FLL, HLL, TRL, and T4L. PCA was performed using the prcomp command in R 4.2.2 (R Core Team 2022). The scatter plot was plotted using the ggplot2 package in R 4.2.2. As species of Diploderma are known to be sexually dimorphic in morphometric measurements (Manthey et al. 2012;Wang et al. 2015Wang et al. , 2018Wang et al. , 2019b, mensural data of each sex are treated separately for analyses.

Molecular analysis
Total genomic DNA for the newly collected specimens was extracted from liver tissues with a standard three step phenol chloroform extraction method (Sambrook et al. 1989). The mitochondrial gene NADH dehydrogenase subunit 2 (ND2) was amplified and sequenced by using published primers (Wang et al. 2019a). PCR and sequencing methods were the same as Liu et al. (2020). All new sequences were edited and manually managed using SeqMan in Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and MEGA 11 (Tamura et al. 2021). Representative species of Pseudocalotes Fitzinger were chosen as outgroups (Liu et al. 2022). Genetic data for 39 species of Diploderma and two species of outgroup taxa were obtained from GenBank (Table 1). The technical computation methods for sequences alignment, best substitution model selection, Bayesian inference (BI) and maximum likelihood (ML) phylogenetic analyses, and genetic divergences calculation were the same as those in Liu et al. (2022).

Results
The obtained sequence alignment is 1028 bp in length. The resulting topologies from BI and ML analyses are consistent (Fig. 2). The specimens from Danba County formed a clade sister to Diploderma flaviceps (Barbour & Dunn, 1919) with strong support; the specimens from Muli County formed a clade sister to the clade consisting of D. daochengense Cai, Zhang, Li, Du, Xie, Hou, Zhou &Jiang, 2022, D. yongshengense Liu, Hou, Rao &Ananjeva, 2022, andD. yulongense (Manthey, Denzer, Hou & Wang, 2012) with strong support; the specimens from Jiulong County formed a clade sister to D. angustelinea Wang, Ren, Wu, Che & Siler, 2020 with strong support; and the specimens from Weixi County formed a clade sister to D. aorun Wang, Jiang, Zheng, Xie, Che & Siler, 2020 with strong support. The minimum uncorrected pairwise distance between the specimens from Danba County and other species of Diploderma is 4.4% (between the population from Danba County and D. flaviceps), the minimum uncorrected pairwise distance between the specimens from Muli County and other species of Diploderma is 5.6% (between the population from Muli County and D. yulongense), the minimum uncorrected pairwise distance between the specimens from Jiulong County and other species of Diploderma is 2.8% (between the population from Jiulong County and D. angustelinea), and the minimum uncorrected pairwise distance between the specimens from Weixi County and other species of Diploderma is 2.9% (between the population from Weixi County and D. aorun), which are all greater than the genetic distance (2.6%) between the two recognized species D. drukdaypo and D. vela (Suppl. material 1).
The first two major principal components from PCA were retained, which accounted for 83.32%-91.12% of the total variances. The scatter plot based on PC1 and PC2 showed that the newly collected specimens and species with closely morphological similarities to them can be segregated in both sexes (Fig. 3).     patterns between dorsolateral stripes on dorsum; (15) a distinct wide black stripe on shoulder fold region on each side; (16) stripes around eye absent or very indistinct; (17) oral cavity, inner lips, and tongue pale flesh colour in life.
Description of holotype. Adult male, SVL 77.0 mm; tail short, TAL 130.0 mm, TAL/SVL 1.69; limbs moderately long, FLL 33.1 mm on left side, FLL/SVL 0.43, HLL 53.5 mm on left side, HLL/SVL 0.69. Head relatively long, HW/HL 0.67, HD/ HW 0.84; snout moderately long, SEL/HL 0.37. Rostral elongated, bordered by five small postrostral scales; dorsal head scales heterogeneous, all strongly keeled; distinct Yshaped ridge on dorsal snout. Nasal oval, separated from first supralabial by two rows of scales on each side; loreals small, keeled; suborbital scale rows 5/4, keeled; canthus rostralis elongated, scales greatly overlapping each other; enlarged, keeled scales forming single lateral ridge from posteroinferior eye to posterosuperior tympanum on each side; tympanum concealed under scales; SL 10/10, smooth. Mental pentagonal; IL 11/11; enlarged chin shields 10/10, smooth, first one connected IL on each side, second one separated from IL by one row of small scales on each side, remaining ones separated from IL by two rows of small scales on each side; ventral head scales heterogeneous in size, anterior and middle ones larger, posterior and lateral ones smaller, all strongly keeled; distinct transverse gular fold present; gular pouch moderately developed.
Distinct shoulder fold present; dorsal body scales heterogeneous in size and shape, all keeled, tip pointing backwards; axillary scales much smaller than remaining dorsals; enlarged dorsal scales forming one intermittent longitudinal row between dorsal crest and dorsolateral stripe on each side, remaining enlarged dorsal scales irregularly scattered on each side of body. Nuchal and dorsal crests discontinuous, separated by a diastema, scales of nuchal and dorsal crests enlarged; strongly erected skin fold under nuchal crest and moderately erected skin fold under dorsal crest; MD 48. Dorsal limb scales strongly keeled, homogeneous on fore-limbs and heterogeneous on hind limbs; F4S 17/16, T4S 21/22. Ventral body scales approximately parallel, homogeneous, all strongly keeled, VN 65. Ventral limb scales parallel, small on upper arms and thighs and larger on forearms and crus, all strongly keeled. Tail scales all strongly keeled, ventral tail scales slightly larger than dorsal tail scales.
Colouration of holotype in life. Dorsal surface of head brownish grey. Two indistinct black transverse bands between orbits on dorsal surface of head. Lateral surfaces of head grey. Two indistinct black stripes from posteroinferior eye to anterior tympanum on each side. Upper lips grey, lower lips white. Oral cavity, inner lips, and tongue with pale flesh colour.
Dorsal surface of body purplish grey. A pale yellow strongly jagged dorsolateral stripe from neck to pelvis on each side of body. A series of dark, hollow, approximately rhomboid-shaped patterns between dorsolateral stripes from neck to base of tail, hollow region pale yellow. A distinct wide black stripe on shoulder fold region on each side. Some irregular black patches below dorsolateral stripe on each side of body, no pale spots on each side of body. Dorsal surfaces of limbs brownish grey with dark transverse bands. Dorsal surface of tail greyish white with distinct dark transverse bands.
Ventral surface of head white with distinct grey reticulated pattern. No gular spot. Ventral surfaces of body and limbs greyish white with no pattern, ventral surface of tail greyish white with indistinct dark transverse bands.
Variations. The variations of metrical characteristics of the type series are provided in Table 2. Other variations are as follows: the transverse bands on the dorsal surface of head and the stripes posteroinferior to the eye are more indistinct in all paratypes; the skin fold under nuchal crest is more weak, no skin fold under dorsal crest, and the dorsolateral stripes are indistinct, pale grey in the female paratype.
Diploderma danbaense sp. nov. is phylogenetically sister to and most similar in morphology characteristic and colouration to D. flaviceps; however, Diploderma danbaense sp. nov. can be differentiated from the latter by having a relatively shorter tail (TAL/SVL 1.61-1. Distribution. This species is currently known only from its type locality in Danba County, Ganzi Prefecture, Sichuan Province, China (Fig. 1).
Natural history. This species is terrestrial, inhabiting the hot-dry valley of the upper Dadu River. There are a few trees and many rocks at the type locality (Fig. 16A, B). All specimens were collected between 11 and 12 a.m. when they were basking on rock piles. Paratypes. KIZ2022058-KIZ2022059, KIZ2022061, three adult females; KIZ2022060, adult male; collecting information all the same as the holotype.
Etymology. The specific epithet refers to Donglang Township, where the new species was discovered.
Diagnosis. Diploderma donglangense sp. nov. can be diagnosed from congeners by a combination of the following morphological characteristics: (1) body size relatively small, SVL 44.9-52.8 mm in adult males, 47.    keeled; distinct Y-shaped ridge on dorsal snout. Nasal oval, separated from first supralabial by two rows of scales on each side; loreals small, keeled; suborbital scale rows 5/4, keeled; canthus rostralis elongated, greatly overlapping with each other; enlarged, keeled scales forming distinct single lateral ridge from posteroinferior eye to posterosuperior tympanum on each side; tympanum concealed under scales; SL 10/9, feebly keeled. Mental pentagonal; IL 11/11; enlarged chin shields 4/4, smooth, first one contacting IL on each side, remaining ones separated from IL by one or two rows of small scales on each side; ventral head scales homogeneous in size, keeled; distinct transverse gular fold present; gular pouch weakly developed.
Distinct shoulder fold present; dorsal body scales heterogeneous in size and shape, all keeled, tip pointing backwards; axillary scales much smaller than remaining dorsals; enlarged dorsal scales forming one continuous longitudinal row between dorsal crest and dorsolateral stripe on each side, remaining enlarged dorsal scales roughly forming three or four longitudinal rows on each side of body. Nuchal and dorsal crests feebly developed; no skin fold under nuchal and dorsal crests; MD 40. Dorsal limb scales strongly keeled, mostly homogeneous, except a few enlarged, conical scales on postaxial thighs; F4S 16/16, T4S 24/23. Ventral body scales approximately parallel, homogeneous, all strongly keeled, VN 63. Ventral limb scales parallel, homogeneous, approximately equal in size to ventrals, all strongly keeled. Tail scales all strongly keeled, ventral tail scales larger than dorsal tail scales.
Colouration of holotype in life. Dorsal surface of head dark brown. Transverse bands on dorsal surface of head indistinct. Lateral surfaces of head brownish grey. A distinct black stripe from posteroinferior eye to tympanum region on each side. Upper lips brownish grey, lower lips white. Oral cavity, inner lips, and tongue pale pink.
Dorsal surface of body dark brown. A creamy yellow moderately jagged dorsolateral stripe on each side of body from occipital region to pelvis. Some indistinct black patterns between two dorsolateral stripes. Some creamy yellow spots scattered below dorsolateral stripe on each side of body. Dorsal surfaces of limbs brown with dark transverse bands. Dorsal surface of tail brownish grey with indistinct dark transverse bands.
Ventral surface of head white. A pale yellow gular spot on posterior central part of ventral head, some short black stripes mostly on anterior and sides of gular spot. Ventral surfaces of body, limbs, and tail white with no pattern.
Variations. The variations of metrical characteristics of the type series are provided in Table 3. Other variations are as follows: the dorsal colour are paler, the patterns between two dorsolateral stripes are more distinct, the transverse bands on the dorsal surface of head are more distinct, and the dorsolateral stripes are indistinct, pale grey or yellow anteriorly and pale grey posteriorly in the female paratypes; the gular spot present in one female paratype and absent in another female paratype; in addition, the short black stripes on ventral surface of head are more indistinct in all paratypes.
Natural history. This species is terrestrial, inhabiting the hot-dry valley of the upper Shuiluo River, which is a tributary of the Jinsha River. There are many thorny shrubs and some rock piles at the type locality (Fig. 16C, D). All specimens were collected between 1 and 3 p.m. when they were basking on rock piles. Paratypes. KIZ2022087 and KIZ2022101, two adult females; KIZ2022099-KIZ2022100, two adult males, collecting information all the same as the holotype.
Etymology. The specific epithet refers to Jiulong County, where the new species was discovered.
Diagnosis. Diploderma jiulongense sp. nov. can be diagnosed from congeners by a combination of the following morphological characteristics: (1)  9/9; enlarged chin shields 4/5, smooth, first one contacting IL on each side, remaining ones separated from IL by two rows of small scales on each side; ventral head scales heterogeneous in size, ones on centre of gular pouch largest, all strongly keeled; distinct transverse gular fold present; gular pouch weakly developed. Distinct shoulder fold present; dorsal body scales heterogeneous in size and shape, all keeled, tip pointing backwards; axillary scales much smaller than remaining dorsals; enlarged dorsal scales roughly forming four longitudinal rows on each side of body. Nuchal and dorsal crests feebly developed, continuous; no skin fold under nuchal and dorsal crest; MD 36. Dorsal limb scales strongly keeled, mostly homogeneous, except a few enlarged, conical scales on postaxial thighs; F4S 18/19, T4S 27/27. Ventral body scales approximately parallel, homogeneous, all strongly keeled, VN 54. Ventral limb scales parallel, small on upper arms and thighs and larger on forearms and crus, all strongly keeled. Tail scales all strongly keeled, ventral tail scales slightly larger than dorsal tail scales.
Colouration of holotype in life. Dorsal surface of head dark grey with no transverse bands. Lateral surfaces of head grey, a greyish white suborbital stripe extending from nasal scale to rictus on each side. Upper lips brownish grey, lower lips greyish white. No radial stripes around eyes. Oral cavity and inner lips pinkish white, tongue pale flesh colour.
Dorsal surface of body brownish black. A narrow, bright yellow, smooth-edged, dorsolateral stripe on each side of body from occipital region to pelvis. Some black inverted triangular patterns between the two dorsolateral stripes. Some bright yellow spots scattered below dorsolateral stripe on each side of body. Dorsal surfaces of limbs dark grey. Dark transverse bands on dorsal surfaces of limbs very indistinct. Dorsal surface of tail brownish grey with some indistinct dark transverse bands. Variations. The variations of metrical characteristics of the type series are provided in Table 4. Other variations are as follows: the dorsal colour is brownish red or brick red, the transverse bands on the limbs are more distinct, the dorsolateral stripes are indistinct, pale grey or yellow anteriorly and pale grey posteriorly, and the gular spot indistinct or absent in the female paratypes.
Distribution. This species is currently known only from its type locality in Yandai Town, Jiulong County, Ganzi Prefecture, Sichuan Province, China (Fig. 1).
Natural history. All specimens were collected between 9 and 12 a.m. in bushes or grass in the Yalong River valley. There are a few trees and many rocks at the type locality (Fig. 16E, F). Description of holotype. Adult male, SVL 55.2 mm; tail moderately long, TAL 104.5 mm, TAL/SVL 1.89; limbs moderately long, FLL 25.9 mm on left side, FLL/ SVL 0.47, HLL 41.7 mm on left, HLL/SVL 0.76. Head relatively robust, HW/HL 0.73, HD/HW 0.87; snout moderately long, SEL/HL 0.36. Rostral elongated, bordered by six small postrostral scales; dorsal head scales heterogeneous, all strongly keeled; distinct Y-shaped ridge on dorsal snout. Nasal oval, separated from first supralabial by single row of scales on each side; loreals small, keeled; suborbital scale rows 4/4, keeled; canthus rostralis elongated, greatly overlapping with each other; enlarged, keeled scales forming distinct single lateral ridge from posteroinferior eye to posterosuperior tympanum on each side; tympanum concealed under scales; SL 9/9, feebly keeled. Mental approximately triangular; IL 10/10; enlarged chin shields 4/4, smooth,  first one contacting IL on each side, second ones separated from IL by one row of small scales on each side, remaining ones separated from IL by two rows of small scales on each side; ventral head scales homogeneous in size, feebly keeled; distinct transverse gular fold present; gular pouch weakly developed.
Distinct shoulder fold present; dorsal body scales heterogeneous in size and shape, all keeled, tip pointing backwards; axillary scales much smaller than remaining dorsals; enlarged dorsal scales forming one intermittent longitudinal row between dorsal crest and dorsolateral stripe on each side, remaining enlarged dorsal scales irregularly scattered on each side of body. Nuchal and dorsal crests feebly developed, continuous; no skin fold under nuchal and dorsal crests; MD 43. Dorsal limb scales strongly Variations. The variations of metrical characteristics of the type series are provided in Table 5. Other variations are as follows: the dorsal colour is paler, there are some dark inverted triangular patterns between the two dorsolateral stripes, the radial stripes around eyes are more indistinct, and the dark stripes on ventral head are more distinct in all paratypes; in addition, the dorsolateral stripes are indistinct, yellowish white in the female paratypes.

Discussion
With a rapidly increasing number of species, the genus Diploderma consists of 42 species in total, whereof nearly half were described in the last five years, and more than one third were described in the last three years (e.g., Cai et al. 2022;Liu et al. 2022;Uetz et al. 2022). Together with the four new species described in this study, we bring the total number of species in this genus to 46.
Among the four new species described in this study, Diploderma jiulongense sp. nov. and Diploderma tachengense sp. nov. have relatively small genetic distances (2.9% and 2.8%) from the closely related species D. angustelinea and D. aorun. However, there are significant morphological differences between Diploderma jiulongense sp. nov. and D. angustelinea and between Diploderma tachengense sp. nov. and D. aorun. Diploderma jiulongense sp. nov. differs from D. angustelinea by having a relatively much longer tail, relatively longer fore-limbs, relatively longer hind limbs, and having smooth edged dorsolateral stripes in males. Diploderma tachengense sp. nov. differs from D. aorun by having an obviously differently coloured gular spot in life, having a relatively shorter tail, and the absence of skin folds under nuchal and dorsal crests in males in life. In addition, the genetic distances between them and their closely related species are both greater than that (2.6%) between the two recognized species D. drukdaypo and D. vela. Although the genetic distance is small between D. drukdaypo and D. vela, they have distinct and stable morphological differences (i.e., relative limb length, relative tail length, and keeling of ventral body scales; Wang et al. 2015Wang et al. , 2019b. Thus, they are regarded as two distinct species each that occupy distinct sections of the Mekong River and live at different elevations (Wang et al. 2021b). Therefore, taking the case of the species pair of D. drukdaypo and D. vela as the calibration, Diploderma jiulongense sp. nov. and Diploderma tachengense sp. nov. also have surpassed intraspecific variation level and reached a specific status.
Another new species described in this study, Diploderma danbaense sp. nov., is at first glance similar in morphology to D. flaviceps. To compare morphological characteristics between Diploderma danbaense sp. nov. and D. flaviceps, we examined 27 topotypic or near-topotypic specimens of D. flaviceps (Suppl. material 2), and found several non-overlapping morphological characteristics (i.e., relative tail length, relative hind limb length, and relative head width) between both taxa (see Table 6). In addition, the genetic distance (4.4%) between Diploderma danbaense sp. nov. and D. flaviceps is approximate to that (4.1%) between D. brevicauda and D. qilin, greater than that (3.2%) between D. formosgulae and D. drukdaypo and that (3.3%) between D. formosgulae and D. vela, and much greater than that (2.6%) between D. drukdaypo and D. vela. Diploderma brevicauda and D. qilin are similar in morphology, according to Wang et al. (2021b), there are only differences in relative tail length, relative hind limb length and oral cavity colour between them, and these differences are considered sufficient to distinguish different species (Wang et al. 2021b).
Allopatric distribution across the Hengduan Mountain Region has been suggested to create reproductive isolation through geographic isolation (Wang et al. 2015(Wang et al. , 2021aDong et al. 2020). Wang et al. (2021b) considered that D. drukdaypo may represent a recently diverged species from the D. vela lineage, adapted to higher-elevated habitats in the upper Mekong River valley, and suggested that further studies are needed to gain a better understanding of gene flow and speciation mechanisms between these two sister species. Although D. formosgulae is morphologically similar and genetically close to D. drukdaypo and D. vela, Wang et al. (2021a) stated that D. formosgulae possesses an allopatric distribution with the latter two species and represents a recently diverged lineage, evolutionarily distinct. Likewise, Diploderma danbaense sp. nov. also possesses an allopatric distribution with D. flaviceps and may represent a recently diverged species, adapted to higher-elevated habitats in the upper Dadu River valley.
To further confirm the validity of these three new species, we conducted detailed surveys in the field to verify whether there is geographical isolation between these three new species and their closely related relatives. At first, the type locality of Diploderma tachengense Table 6. Comparison between Diploderma danbaense sp. nov. and D. flaviceps. For morphological abbreviations see the Materials and methods. Detailed measurements and pholidosis data for each examined specimen of D. flaviceps are given in Suppl. material 2. sp. nov. (at 2180 m elevation) is approximately 100 km downstream along the Jinsha River from the type locality of D. aorun (at 2198 m elevation). Although the altitudinal ranges are similar, their habitats are completely different, one is a hot-dry valley of the Jinsha River, the other is a distant forested region. Therefore, there is a clear geographical and ecological isolation between the populations of Diploderma tachengense sp. nov. and D. aorun. Further, the type locality of Diploderma jiulongense sp. nov. (at 1680 m elevation) is approximately 130 km downstream along the Yalong River from the type locality of D. angustelinea (at 2017 m elevation). Although the linear distance between the two localities is short, there is a branch of the Daxue Mountain, with the highest peak at approximately 5000 m elevation, between them, serving as a clear geographic barrier. In addition, there is another population of Diploderma (D. cf. daochengense) occupying part valley section of the Yalong River, which bypasses the branch of the Daxue Mountain, between the type localities of Diploderma jiulongense sp. nov. and D. angustelinea. At last, the type locality of Diploderma danbaense sp. nov. (at 2020 m elevation) is approximately 150 km upstream along the Dadu River from the type locality of D. flaviceps (at 1300 m elevation). Although there is no obvious geographical barrier between the two localities except for the altitudinal difference, we have not found any transitional type individuals between Diploderma danbaense sp. nov. and D. flaviceps, nor have we found Diploderma danbaense sp. nov. and D. flaviceps occurring in sympatry. In addition, the relatively great genetic distance (4.4%) between Diploderma danbaense sp. nov. and D. flaviceps also supports an isolation process. A similar situation occurs between the two recognized closely related species D. panlong and D. swild. Through our observation, we found that these two species are similar in morphology and colouration, their habitats and natural history are also similar, as both inhabit forests or forest margins and both are more arboreal than terrestrial, and there is no obvious geographical barrier between them. However, these two species have not been found in sympatry, and there is a great genetic distance between them. We speculate that some historical reasons may have led to the isolation of different populations, thus, they lost genetic exchange and differentiated into different species.
Finally, the mitochondrial ND2 gene is considered to be able to better distinguish different species and has been widely used in phylogenetic analyses of Agamidae (i.e., Zug et al. 2006;Grismer et al. 2016;Ambekar et al. 2020;Liu et al. 2021;Wang et al. 2022a), and the phenomenon of different species with small genetic distances in the ND2 gene is well known from other genera of Agamidae, such as in the genus Sitana Cuvier, where a genetic distance of 3-4% in the ND2 gene is considered sufficient to distinguish different species (Ambekar et al. 2020). This supports our taxonomic actions presented herein, viz. that different populations with relatively small genetic distances in the ND2 gene can represent different species in the Agamidae.