A New Species of Diploderma (Squamata, Agamidae) from the Valley of Dadu River in Sichuan Province, with a Redescription of Topotypes of D. splendidum from Hubei Province, China

Simple Summary The genus Diploderma Hallowell, 1861 (Squamata, Agamidae), currently consists of 46 species, which are distributed across East Asia (inclusive of Japan) and the northern part of the Indochinese Peninsula, primarily inhabiting dry valley regions in Western China. This article introduces a novel species of Diploderma based on its unique morphological features and molecular evidence, discovered in the lower valley of the Dadu River in Sichuan Province, Western China. Phylogenetic analysis using ND2 data suggests that this new taxon is distinct from its congeners. Morphologically, the new species can be differentiated from other Diploderma species by an assemblage of 46 specific characteristics. Principal component analysis (PCA) further demonstrates that this new species is clearly distinguishable from its closest relative, D. splendidum. Given these multiple lines of evidence, we describe this species from the lower Dadu River valley as a newly identified species, D. daduense sp. nov. This discovery brings the total number of recognized species within the genus Diploderma to 47. Abstract This study describes a novel species of Diploderma (Squamata, Agamidae) from the lower valley of the Dadu River of the Sichuan Province of Western China based on its distinct morphological features and molecular evidence. D. daduense sp. nov. can be distinguished from its congeners by its tympanum concealed; head mainly green-yellow, supplemented by black; skin folds under the nuchal and dorsal crest obviously present in adult males only, its vertebral crest discontinuous between nuchal and dorsal sections with a distinct gap; transverse gular fold present but not obvious in some individuals; gular spot absent in both sexes; dorsolateral stripes green-yellow anteriorly, cyan in the center and blurry off-white posteriorly in adult males, the upper edge of dorsolateral stripes strongly jagged in adult males; no radial stripes around the eyes; inner-lip coloration smoky-white, and the coloration of the tongue and oral cavity as a light-flesh color in life; bright green-yellow transverse stripes on dorsal body in males; black patches are evenly distributed along the vertebral line between the dorsolateral stripes from the neck to the base of the tail in males; beech-brown or gray-brown line along the vertebral line with heart-shaped or diamond-shaped black patches on the dorsal body in females; and supratemporals fewer than four on at least one side. The phylogenetic tree based on mitochondrial ND2 sequences indicates that D. daduense sp. nov. forms an independent clade with strong support 1/100 in ML bootstrap/Bayesian posterior probability and is the sister group to D. splendidum. At the inter-species level, the p-distance is at least 6.95%, further confirming that an independent species had been identified. Our work raises the number of species within the genus Diploderma to 47.


Introduction
Diploderma Hallowell, 1861, is one of the most diverse genera within the family Agamidae, comprising at least 46 recognized species, distributed throughout East Asia (including Japan) and the northern part of the Indochinese Peninsula [1,2].Extensive field surveys and the application of DNA barcoding technology have significantly advanced our understanding of species diversity within the Diploderma genus.
In molecular systematics, these species form two distinct major clades on the phylogenetic tree [1].One clade encompasses species distributed in the central or southern parts of the Hengduan Mountains, whereas the other clade primarily consists of species found in the eastern part of the Hengduan Mountains, extending their range to Taiwan Island and Japan.The minimum uncorrected pairwise distance among these species is significantly greater than the genetic distance between the two recognized species D. drukdaypo and D. vela, which stands at 2.6% [1].
Most Diploderma species exhibit prominent gular spots, and variations in the coloration of these spots play a crucial feature in species classification and identification [3].Additionally, the color and pattern of dorsolateral stripes, as well as the presence or absence of radiating marks around the eyes, contribute to distinct traits for differentiation [4].Diploderma species display typical sexual dimorphism in morphometric measurements, with males generally being more vividly colored than females [1,5].
The majority of Diploderma species are distributed along dry valley regions, particularly in the valleys of the Jinsha, Yalong, Nu, and Lancang rivers, displaying remarkable diversity and endemism across a wide altitudinal range [3].Notably, numerous Diploderma species occupy and adapt to different stretches of river valleys within the Hengduan Mountains.In 2022, Shi et al. conducted a habitat preference and morphological differentiation study on nine Diploderma species along the Nu, Lancang, and Jinsha rivers, discovering that lizards from different habitat types exhibit specific preferences for habitat factors and have undergone relatively conspicuous differentiation in body size and locomotion characteristics between shrub-type and forest-type lizards [6].
However, the dry valleys of the Dadu River have received less attention.The Dadu River is a crucial tributary of the Yangtze River, joining the Min River at Leshan City before merging with the Jinsha River to form the Yangtze River at Yibin City.To date, three species of Diploderma (Figure 1) have been identified in the Dadu River Valley [1]: D. danbaense Liu, Hou, Ananjeva, and Rao, 2023 and D. flaviceps (Barbour & Dunn, 1919) above Luding County, while the area between Luding County and Leshan City is believed to harbor D. splendidum (Barbour & Dunn, 1919).D. splendidum's distribution spans across the four provinces (Yunnan, Sichuan, Hubei, and Chongqing) [7], where there are significant variations in climate and terrain.The downstream reaches of the Dadu River are more than 700 km away from Yichang City, the type locality of D. splendidum, and the difference in climate conditions between these two areas is substantial.Populations of D. splendidum in these two regions also exhibit morphological differences.
In the period from 2018 to 2022, we conducted a series of field surveys in and around the Dadu River Valley and the gorge vicinity of Yichang City, during which we collected a series of specimens initially identified as Diploderma splendidum.Following molecular phylogenetic analyses and morphological comparisons, it became evident that the population from Dadu River Valley exceeds the threshold of intraspecific variation.Consequently, we describe one new species of Diploderma herein.

Sampling
Fieldwork was conducted in the vicinity of the Dadu River in Sichuan Province between June 2018 and June 2021 by Bo Cai, Yayong Wu, Ling Li, and Dong Liang and in the area surrounding Yichang City, Hubei Province, in August 2022 by Bo Cai.Live animals were photographed to document their color patterns prior to euthanasia.Euthanasia was performed through the intracelomic injection of 250 mg/kg of 1% MS-222 (Chengdu, China) for anesthesia, followed by intracelomic injection of 75% alcohol (Chengdu, China).Liver tissue samples were collected and stored in 95% ethanol for molecular analyses, and specimens were preserved in 75% ethanol.
Specimens were deposited at the Museum of Herpetology, Chengdu Institute of Biology, Chinese Academy of Sciences (CIB), the Museum of Yibin Key Laboratory of Animal Diversity and Ecological Conservation, Yibin University (YBU), and the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Morphological Analysis
Morphological data of recognized Diploderma species were obtained from the examination of museum specimens (Table S1), including CIB, YBU, and KIZ, and from the literature by Zhao et al. (1999) and Liu et al. (2023) [1,4].
Measurements were taken mainly following Zhao et al. (1999) [4] to the nearest 1 mm using a steel ruler for snout-vent length and tail length and to the nearest 0.1 mm using a digital caliper for other relatively short measurements.In total, 11 measurements were measured: the snout-vent length (SVL) from the snout tip to the anterior edge of the vent; tail length (TAL) from the anterior edge of the vent to the tip of the tail; head width (HW) between the widest points in the temporal region, anterior to the tympanum; head length (HL) from the tip of the snout to the right angle of the jaw; head depth (HD) at the temporal region of the head; snout-eye length (SEL) from the tip of the snout to the anterior margin of the orbit; length of the tallest nuchal crest (TNC) from the bottom to the tip of the tallest nuchal crest; foreleg length (FLL) from the axilla to the tip of finger IV, excluding the claw, measured with the limb straightened; hindleg length (HLL) from the groin to the tip of toe IV, excluding the claw, measured with the limb straightened; trunk length (TRL) from the armpit to the groin; and toe IV length (T4L) from the tip of toe IV to the base between toe III and IV, excluding the claw.To avoid the effects of allometry, measurement data from larvae and subadult individuals were excluded from morphological comparisons.
The definitions of morphological characteristics and the counting methods also mainly followed Zhao et al. (1999) and Liu et al. (2023) [1,4] as follows: the supralabial scale count (SL) from the rostral to the corner of the mouth; nasal-supralabial scale rows (NSL) between the first supralabial and the nasal; infralabials (ILs) from the mental to the corner of the mouth; ventrals (VNs) counted in a straight line along the medial axis between the transverse gular fold and the anterior edge of the cloaca; gulars (GUs) counted in a straight line along the medial axis between and excluding the mental and transverse gular fold; middorsal crest scales (MD) counted longitudinally from the first nuchal crest to the scale above the cloaca; finger IV subdigital lamellae (F4S) from the base between finger III and IV to the tip of finger IV, excluding the claw; toe IV subdigital lamellae (T4S) from the base between toe III and IV to the tip of toe IV, excluding the claw; radial stripes below the eyes (RSBE) absent or present; gular fold state (GF) absent or present; gular pouch state (GP) absent or present; gular spot color (GSC); tympanum state (TS) concealed with scales or not; skin fold under the nuchal crest (SFNC) absent or present; nuchal crest state (NC) strongly erected or not; skin fold under the dorsal crest (SFDC) absent or present; shape of dorsolateral stripes (SDS) in males, either smooth edged or jagged; ventral scale state (VSS) absent or present; hindlimbs adpressed forward (HAF) to the reaching area; and supratemporals (ST) enlarged, modified temporal scales.Coloration descriptions are used terminology and codes in the RGB (red, green, blue) color model [8].Data on coloration and ornamentation were collected from live specimens in both unstressed and stressed states, including the following: inner-lip coloration (ILC); the coloration of the oral cavity (CO): defined as the background coloration of the anterior roof and sides of the mouth, excluding the posterior palate and deep throat; the coloration of the tongue (CTG): defined as the coloration of the tongue; the coloration of the dorsolateral stripes (CDS): defined as the background coloration of the dorsolateral stripes; ventrolateral body coloration (VLBC); and ventral body coloration (VBC).
A principal component analysis (PCA) was utilized to assess whether newly collected specimens and species occupied distinct positions in morphospace and to confirm the consistency of the results with the species descriptions provided by molecular phylogenetic analyses.Characters used in the PCA were mensural data from SVL, TAL, HL, HW, HD, SEL, FLL, HLL, T4L, and TRL, with morphological data for D. flaviceps taken from Liu et al. 2023 [1].The PCA was performed using the prcomp command in R version 4.3.2[9] and scatterplotting was conducted with the R package ggplot2 3.4.4[10].Considering that sexual dimorphism is acknowledged in morphometric measurements for species within the genus Diploderma [1,5,8], mensural data for each sex were analyzed separately.
The acquired nucleotide sequences were initially subjected to forward and reverse strand proofreading and editing using SeqManin 7.1.0.44 the DNAstar 7.1 software package (DNAStar Inc., Madison, WI, USA) [12].There were 45 novel sequences of ND2 obtained in this work, and a total of 92 ND2 sequences of 45 Diploderma species were downloaded from GenBank for phylogenetic analyses.Corresponding sequences of Pseudocalotes brevipes (AF128502), Bronchocela cristatella (KR053114), and Laodracon carsticola (OR544068) were downloaded from GenBank and used as outgroups (Table 1).Sequences were aligned in MEGA 11 [13] using ClustalW [14] with default settings.Phylogenetic analyses were conducted using Bayesian inference (BI) and maximum likelihood (ML) methods implemented in PhyloSuite v1.2.3 [15] and MEGA 11, respectively.Before the phylogenetic analyses, the best evolutionary model was conducted and chosen under the Bayesian inference criteria (BIC) using ModelFinder [16].The GTR + I + G4 model was selected as the best model for the mitochondrial gene.In the BI analyses, two runs were performed simultaneously with four Markov chains.The chains were run for 10,000,000 generations and sampled every 1000 generations.The first 25% of the sampled trees were discarded as burn-in, and then the remaining trees were used to estimate Bayesian posterior probabilities (BPPs); nodes with BPP values of 0.95 and higher were considered well supported [17,18].In the ML analyses, 1000 bootstrap pseudoreplicates via the ultrafast bootstrap (UFB) approximation algorithm were used to construct a final consensus tree; nodes with UFB values of 95% and above were considered significantly supported [19].Finally, uncorrected genetic pairwise distances (p-distances) for ND2 were calculated using default parameters in MEGA 11 [13].

Phylogenetic Analyses
The ND2 sequence alignments were 974 bp in length, and two consistent topologies of phylogenetic trees were obtained from BI and ML analyses, with all analyzed species of Diploderma forming a strongly supported monophyletic group.We further identified the presence of six unique subclades within this group.Among these, subclades I through V coalesced into a larger clade, which, in conjunction with the VI subclade, constituted the overall monophyletic group (Figure 2).The newly collected samples from the lower valley of the Dadu River population, Leibo population, Yibin population, and Yichang population are clustered into four distinct lineages in the context of subclade I, respectively, and are well supported.Specifically, clades from Yibin and Leibo clustered together first, then grouped with the population from the lower valley of the Dadu River, and finally aggregated with the population from the population from Yichang.
Uncorrected mean genetic distances ranged from approximately 1.44% to 22.69% across all samples, whereas the genetic distance between the population from the lower valley of the Dadu River population and other congeners is at least 6.95%, between the population from Yibin is 6.99%, and between the population from Leibo is 7.29%, which are much higher than the shortest genetic distance of the recognized Diploderma species (2.6% between D. drukdaypo and D. vela) (Table S2).However, the genetic distance between D. daochengense and D. yongshengense is only 1.44%, which may imply that one of these two species is a subspecies of the other, and we will conduct further research in the future.The phylogenetic tree of the genus Diploderma inferred from the mitochondrial ND2 (974 bp).ML bootstrap support/Bayesian posterior probability support is denoted above each node.The maximum parsimony strict consensus tree for the Diploderma species is inferred from ND2 gene fragments by using IQ-TREE v2.2.0 [20]; the ND2 gene majority-rule consensus tree for the Diploderma species is inferred from partitioned Bayesian analyses by using MrBayes v3.2.7a [21].In view of the unique clades of the Yibin and Leibo populations, sp.1 and sp.2 are used instead, respectively.
Uncorrected mean genetic distances ranged from approximately 1.44% to 22.69% across all samples, whereas the genetic distance between the population from the lower valley of the Dadu River population and other congeners is at least 6.95%, between the population from Yibin is 6.99%, and between the population from Leibo is 7.29%, which are much higher than the shortest genetic distance of the recognized Diploderma species (2.6% between D. drukdaypo and D. vela) (Table S2).However, the genetic distance between D. daochengense and D. yongshengense is only 1.44%, which may imply that one of these two species is a subspecies of the other, and we will conduct further research in the future.The maximum parsimony strict consensus tree for the Diploderma species is inferred from ND2 gene fragments by using IQ-TREE v2.2.0 [20]; the ND2 gene majority-rule consensus tree for the Diploderma species is inferred from partitioned Bayesian analyses by using MrBayes v3.2.7a [21].In view of the unique clades of the Yibin and Leibo populations, sp.1 and sp.2 are used instead, respectively.

Morphological Analyses
From the results of the principal component analyses (PCA), it can be seen that the lower valley of the Dadu River population overlaps only slightly with the Yichang City population, and they are completely separated from D. flaviceps in morphospace (Figure 3).For the morphometric data of males, the cumulative proportions of the first two PCs accounted for 90.77% of the variance, and the eigenvalues were all greater than one.For the first two PCs, they load the most heavily on SVL, HL, HD, and HW (Table S3).For the female morphometric data, the cumulative proportion of the first two PCs accounted for 84.40% of the variance, and the eigenvalues were all greater than 1, as for males.For the first two PCs, they load the most heavily on HLL, SVL, HD, and HW (Table S4).

Morphological Analyses
From the results of the principal component analyses (PCA), it can be seen that the lower valley of the Dadu River population overlaps only slightly with the Yichang City population, and they are completely separated from D. flaviceps in morphospace (Figure 3).For the morphometric data of males, the cumulative proportions of the first two PCs accounted for 90.77% of the variance, and the eigenvalues were all greater than one.For the first two PCs, they load the most heavily on SVL, HL, HD, and HW (Table S3).For the female morphometric data, the cumulative proportion of the first two PCs accounted for 84.40% of the variance, and the eigenvalues were all greater than 1, as for males.For the first two PCs, they load the most heavily on HLL, SVL, HD, and HW (Table S4).In addition, the population from the lower valley of the Dadu River also has its own unique morphological characteristics (Table 2).It showed different morphological characteristics from sister species D. splendidum: a bigger body size and heard in adult males, gular spot absence in both sexes; the upper edge of dorsolateral stripes is strongly jagged and lower edge smooth in adult males; and so on.We will elaborate on this in more detail in the following diagnosis.In addition, the population from the lower valley of the Dadu River also has its own unique morphological characteristics (Table 2).It showed different morphological characteristics from sister species D. splendidum: a bigger body size and heard in adult males, gular spot absence in both sexes; the upper edge of dorsolateral stripes is strongly jagged and lower edge smooth in adult males; and so on.We will elaborate on this in more detail in the following diagnosis.

Taxonomy Accounts
Both the molecular phylogenetic relationships and the uncorrected pairwise distance inferred from ND2, and the integration of the differences from morphological data and the morphospace, suggest that the lower valley of the Dadu River population represents one new species.We described it below.
3.3.1.Diploderma splendidum (Barbour and Dunn, 1919) Japalura splendida Barbour and Dunn, 1919 [22] Holotype.USNM 35522, an adult male "from the gorge of the Yangtze River near Ichang (=Yichang City), Hupeh (=Hubei Province), central China", collected by E. Blackwelder.After reviewing the original documents and the old map involved, we believe that this gorge should be the Lower Gorge section of Xiling Gorge in Yiling District, Yichang City, China.

Description of topotypes
Coloration of topotypes in life (Figures 4 and 5A,B).In life, adult males have a yellow (255, 255, 0) gular spot, which is consistent with Barbour and Dunn (1919) [22] and Xu et al. (2023) [23], green-yellow to cyan (0, 255, 255) dorsolateral stripes, and adult females have green-yellow (173, 255, 47) dorsolateral stripes.No radial stripes around the eyes; the inner-lip coloration is smoky-white (245, 245, 245), and the coloration of the tongue and oral cavity is a light-flesh color (239,205,197) in life; green-yellow transverse stripes on the dorsal body, near-square-shaped black patches are evenly distributed along the vertebral line between the dorsolateral stripes from the neck to the base of the tail in both sexes.When stressed, the color of ventral head turns dim-gray (105, 105, 105) with slightly larger scales turning white (255, 255, 255), the ventral body coloration turning dim-gray, and the ventrolateral body turning black (0, 0, 0).
Distribution and natural history of topotypes.According to the nearby tributaries of the Yangtze River and the topography, the Xiling Gorge is divided into four sections from upstream to downstream: the Xiangxi Wide Canyon, Upper Wide Canyon, Miaonan Wide Canyon, and the Lower Gorge.Our sampling covers almost all of Xiling Gorge, which is near the Yangtze River, elevation 99-669 m.This population of Diploderma splendidum is terrestrial, inhabiting the central subtropical evergreen broad-leaved forest of the Xiling Gorge and its surrounding hills (Figure 1).
These lizards are commonly found on crevice-filled cliffs and on rock piles or soil walls at forest edges (Figure 4).All specimens were collected between 08:00 and 17:00 h in August.
Diploderma splendidum is oviparous.Specimens from Yichang City obtained in August had 2-6 eggs.Distribution and natural history of topotypes.According to the nearby tributaries of the Yangtze River and the topography, the Xiling Gorge is divided into four sections from upstream to downstream: the Xiangxi Wide Canyon, Upper Wide Canyon, Miaonan Wide Canyon, and the Lower Gorge.Our sampling covers almost all of Xiling Gorge, which is near the Yangtze River, elevation 99-669 m.This population of Diploderma splendidum is terrestrial, inhabiting the central subtropical evergreen broad-leaved forest of the Xiling Gorge and its surrounding hills (Figure 1).
These lizards are commonly found on crevice-filled cliffs and on rock piles or soil walls at forest edges (Figure 4).All specimens were collected between 08:00 and 17:00 h in August.
Diploderma splendidum is oviparous.Specimens from Yichang City obtained in August had 2-6 eggs.
Description of Holotype.Adult male, body large-sized, SVL 93.5 mm; tail long, TAL 245.2 mm; head longer (30.0 mm) than wide (22.6 mm); head depth 15.1 mm; length of tallest nuchal crest 2.5 mm; snout-eye length 11.1 mm; foreleg length 42.4 mm; hindleg length 72.0 mm; toe IV length 18.3 mm; trunk length 49.0 mm; snout moderately long, SEL/HL 0.37.Rostral flat, bordered by 5 small postrostral scales; dorsal head scales heterogeneous in size, most of them keeled.Number of scales between eyes 17; nasal scale approximately oval; internasals 7; loreals 6/6, small, unkeeled; supralabials 8/8, infralabial scales 8/9, supratemporals 4/3, and middorsal crest scales 45, keeled; gular scales 33 and ventral scales 64; finger IV subdigital lamellae 21/23; toe IV subdigital lamellae 29/31; nasalsupralabial scale rows 1/1; suborbital scale rows 2/2.Below subocular, there are 3 parallel rows of scales, and first row extends from front of anterior nasal to upper post-rictus, second row extends from postrostrals to upper post-rictus, and third row is supralabials; transverse gular fold present; gular pouch present in life; tympanum concealed, covered with small keeled scales; well-developed skin fold under nuchal crest present, vertebral crest discontinuous between nuchal and dorsal sections with gap; dorsal and ventral scales distinctively keeled exclude scales around eyes and lips; dorsal scales of head, trunk, limbs, and tail heterogeneous in size, ventral scales of head homogeneous in size with few larger scales, ventral scales of trunk homogeneous in size; fold present in front of shoulder; fourth toe with claw reaching between tympanum and eye when hindlimbs adpressed forward.Upper edges of dorsolateral stripes are strongly jagged, but lower edge is relatively smooth.5C,D and 6).The dorsal head is mainly greenyellow, with two black transverse stripes connecting the left and right supraoculars, black spots on the frontal, and black and green-yellow markings on the anterodorsal edge.The green-yellow ground color of the head is maculated with black.There are four stripes around the upper eye except the subocular regions on each side, with a thick black stripe extending from the posterior nasal through the lower palpebrals to the temporal region.The first subocular scale row is white in the middle and green-yellow at both ends, second row black.The supralabials and infralabials are white, turning gray-brown when stressed.Below are infralabials with beech-brown lines.Gular spot is absent.The ventral scales of the head are white with gray-brown lines, turning gray-brown with beech-brown lines and scattered with white spots when stressed.The inner-lip coloration is smoky-white, and the coloration of the tongue and oral cavity is a light-flesh color.

Coloration of Holotype in life (Figures
with small keeled scales; well-developed skin fold under nuchal crest present, vertebral crest discontinuous between nuchal and dorsal sections with gap; dorsal and ventral scales distinctively keeled exclude scales around eyes and lips; dorsal scales of head, trunk, limbs, and tail heterogeneous in size, ventral scales of head homogeneous in size with few larger scales, ventral scales of trunk homogeneous in size; fold present in front of shoulder; fourth toe with claw reaching between tympanum and eye when hindlimbs adpressed forward.Upper edges of dorsolateral stripes are strongly jagged, but lower edge is relatively smooth. Coloration of Holotype in life (Figures 5C,D and 6).The dorsal head is mainly greenyellow, with two black transverse stripes connecting the left and right supraoculars, black spots on the frontal, and black and green-yellow markings on the anterodorsal edge.The green-yellow ground color of the head is maculated with black.There are four stripes around the upper eye except the subocular regions on each side, with a thick black stripe extending from the posterior nasal through the lower palpebrals to the temporal region.The first subocular scale row is white in the middle and green-yellow at both ends, second row black.The supralabials and infralabials are white, turning gray-brown when stressed.Below are infralabials with beech-brown lines.Gular spot is absent.The ventral scales of the head are white with gray-brown lines, turning gray-brown with beech-brown lines and scattered with white spots when stressed.The inner-lip coloration is smoky-white, and the coloration of the tongue and oral cavity is a light-flesh color.Five dorsal patches are black with small green-yellow spots; dorsolateral stripes are green-yellow anteriorly, cyan in the center, and blurry off-white posteriorly; the upper edge of dorsolateral stripes is strongly jagged, and the lower edge is relatively smooth.The ventrolateral body is light-gray (211, 211, 211), gray-brown to black with white or graybrown spots; the venter is white but off-white to light-gray or gray-brown when stressed; the ventral sides of the limbs are the same as the venter.The dorsal sides of the limbs are gray-brown, interspersed with green (0, 128, 0) and black.The green transverse patterns of the forelimbs are clear, and those of the hindlimbs are blurred.The tail has 15 gray rings.
Variation.Diploderma daduense sp.nov. is sexually dimorphic: (1) males are larger than females, SVL 74.Distribution and Natural History.Diploderma daduense sp.nov. is terrestrial, inhabiting the central subtropical evergreen broad-leaved forest of the lower valley of the Dadu River and its surrounding hills (Figure 1), which is distributed in Shimian County, Jiulong County, Hanyuan County, Ganluo County, Ebian County, Jinkouhe District, Emeishan City, and other places in Sichuan Province, China, with the elevation 574-2063 m.
Compared with the warm-dry habitat of Diploderma flaviceps and D. danbaense in the upper reaches of the Dadu River, part of the habitat of D. daduense sp.nov is located in the West China Rain Screen Area, which is wetter and cooler, and the Fraction Vegetation Coverage is higher.Only near Hanyuan County in the central part of the distribution of D. daduense, the habitat is a warm-dry valley.D. daduense sp.nov is commonly found on tree branches, shrublands, crevice-filled rock piles, or soil walls at forest edges (Figure 6).All specimens were collected between 09:00 and 18:00 h in June and July.
Diploderma daduense sp.nov is oviparous, mating in April to May every year, and laying eggs in June to July [24].We found this species to contain 2-7 eggs.The smallest female CIB-201806035 is only 52.6 mm in snout-vent length and has already conceived 2 eggs.Gao et al. (2004) studied the hatching of 6 eggs (12.6-13.8[average 13.10] mm long and 8.0-9.0 [average 8.56] mm wide), found that the incubation period lasted approximately 49 days, from 20 June 2001 to 7 August 2001, and the snout-vent length of the four hatchlings was 23-25 (24.5) mm, and the tail length was 49-52 (51.3) mm [24].We found this species to prey on larvae and adults of arthropods such as insects and spiders.

Discussion
The genus Diploderma boasts significant diversity, yet many of its species are characterized with insufficiently detailed distribution records and limited genetic data derived from multiple specimens.The discovery of D. daduense sp.nov.has notably augmented our comprehension of species within the genus Diploderma, providing a more comprehensive perspective on morphological variations, distribution limits, and genetic diversity.D. daduense sp.nov.exemplifies a new crucial role played by the Hengduan Mountains in the process of speciation.This finding not only aids in the investigation of the evolutionary relationships between the middle and upper reaches of the Yangtze River but also sheds light on the geological history of the Dadu River Valley.Moreover, it serves as a cornerstone for future studies in species ecology and related fields.
In genetics, sequences of Diploderma daduense sp.nov.have been resolved into three clades.These three clades correspond to geographical areas within the lower reaches of Luding County: The upper section, encompassing both Shimian and Jiulong, forms one clade; the middle segment around Hanyuan constitutes a separate clade; and the lower segment, extending from parts of Hanyuan downwards to Emeishan and Jinkouhe, makes up the third clade.Of note, among these specimens, there is one from Hanyuan that has been identified as belonging to the Jinkouhe clade.In contrast to the tight association between genetic information and distribution in D. daduense sp.nov., such a relationship is more dispersed in D. splendidum.The molecular sequence of the specimen from Jiangyou City and Fujian Province grouped with that of the topotypical D. splendidum specimens led Xu et al. (2023) to hypothesize that the Fujian population may be an introduced species resulting from wildlife trade and Buddhist release activities [23], implying that the Jiangyou population could have also been sourced in this manner.However, definitive confirmation necessitates the collection of additional samples and the implementation of microsatellite analysis.In contrast, D. daduense sp.nov. is currently minimally affected by the pet trade.
In the PCA, there are significant differences between the Diploderma splendidum group and D. flaviceps, while the morphospace for D. splendidum from Yichang also exhibited considerable divergence compared to those from D. daduense sp.nov.Unfortunately, we lack individual morphological data for sp.1 and sp.2, which means that their relationships in the PCA context with respect to D. daduense sp.nov.remain to be further investigated.
In climatological and geographical terms, the unique terrain of the Hengduan Mountains has given rise to a distinct climatic-geographical unit known as dry valleys.These valleys exhibit higher temperatures and lower humidity compared to their surrounding areas.Based on differences in temperature and humidity, the arid valleys in southwestern China can be classified into three categories: hot-dry valleys, warm-dry valleys, and temperate-dry valleys [25].The local climatic-geographical units along the Dadu River are notably associated with species from Diploderma [25][26][27][28].The distribution area for D. danbaense is primarily situated in "semi-arid" warm-dry valleys near Danba, while D. flaviceps inhabits "semi-arid but slightly more humid" warm-dry valleys close to Luding.The habitat range for D. daduense sp.nov.extends across "semi-arid but humid" warm-dry valleys and downstream into the West China Rain Zone.The species sp.1 in Leibo is native to the hot-dry valley of the Jinsha River.The city of Yibin where sp.2 is found belongs to a subtropical monsoon climate zone with moderate moisture, which also exhibits characteristics of a South Asian tropical climate in its low hills and valleys [29].The Yiling District, where the type locality of D. splendidum is located, sits within a mid-subtropical monsoon climate region featuring distinct seasons, mild climates, and moderate precipitation [30].These instances appear to demonstrate that different geographical and climatic environments give rise to distinct species.
In the species distribution boundaries, Diploderma daduense sp.nov., similarly to other Diploderma species within the Hengduan Mountains, is characterized as a valley-dwelling species.The valley-type distribution of D. daduense is bounded to the west by Mount Daxue, to the south by Mount Xiaoxiangling and Mount Daliang, to the north by Mount Daxiangling, and to the east by the Sichuan Basin.The upper portion of the valley-type distribution for D. daduense directly borders, yet does not overlap with, the lower reaches for D. flaviceps, where their western distribution boundary is demarcated by the Dagou River at the confluence of Luding and Shimian counties, while the eastern distribution boundary is estimated be situated about 20 km away from the Dagou River mouth, based on existing specimens.To date, no evidence of overlapping distributions has been found between D. daduense and D. flaviceps nor have any hybrid individuals been morphologically identified.The Yangtze River potentially acts as a separator between D. daduense and sp.1 found in Yibin, which is currently known only from the southern bank of the Yangtze River in Yibin City.Moreover, Mount Daliang serves as a barrier preventing contact between D. daduense and sp.2 found in Leibo County.As a result, geographically, D. daduense is isolated from D. flaviceps and other members of the D. splendidum species group, with no currently documented instances of overlapping distribution areas.
As a major tributary of the Yangtze River, the Dadu River boasts a complex terrain that renders it highly susceptible to geological disasters within China.Thousands of potential geological hazards have been identified in this region, predominantly landslides and debris flows [31].Despite this, the valley accommodates at least five large hydropower stations.The construction of dams and reservoir impoundment activities have exacerbated ecological issues such as soil erosion, an increased frequency of landslides, the inundation of riparian vegetation, and heightened population density around reservoir areas [31].Both naturally occurring and human-induced geological threats pose risks of extensive habitat destruction, potentially impacting the survival of Diploderma daduense sp.nov.
Despite the array of threats confronting Diploderma daduense sp.nov., its current nonendangered status might stem from its extensive distribution range and the prevalence of numerous tributaries within its habitat.However, it remains crucial to diligently monitor the population dynamics of this species to ensure its continued conservation and protection.

Conclusions
The discovery and formal description of Diploderma daduense sp.nov.significantly contributes to our understanding of the diverse genus Diploderma (Squamata, Agamidae), now comprising 47 species.Found in the lower Dadu River Valley, Sichuan Province, West-ern China, this novel species showcases a unique combination of morphological attributes and distinct genetic divergence.Its characterization enhances the knowledge of Diploderma species, particularly regarding detailed distribution patterns, morphological variation, and genetic diversity, addressing the paucity of such data for many existing members of the genus.D. daduense sp.nov.underscores the critical role of the Hengduan Mountains in speciation processes and provides valuable insights into the evolutionary relationships between the middle and upper reaches of the Yangtze River, as well as the geological history of the Dadu River Valley.This groundbreaking finding sets a solid foundation for future ecological, evolutionary, and conservation research within the Diploderma genus and associated disciplines.
Other examined specimens.
14, x FOR PEER REVIEW 3 of 22 population from Dadu River Valley exceeds the threshold of intraspecific variation.Consequently, we describe one new species of Diploderma herein.

Figure 1 .
Figure 1.A map showing the locations of Diploderma specimens in the Dadu River Valley (B) and the locations of D. splendidum topotypes in Yichang (A,C).The red five-pointed star is D. daduense sp.nov., the green triangle is D. flaviceps, the yellow circle is D. splendidum, and the blue square is D. danbaense.The dotted coil represents the approximate distribution range of species in and around the Dadu Valley, and the dotted line represents the approximate distribution boundary of these species.The elevation data were obtained from Geospatial Data Cloud (2022).

Figure 1 .
Figure 1.A map showing the locations of Diploderma specimens in the Dadu River Valley (B) and the locations of D. splendidum topotypes in Yichang (A,C).The red five-pointed star is D. daduense sp.nov., the green triangle is D. flaviceps, the yellow circle is D. splendidum, and the blue square is D. danbaense.The dotted coil represents the approximate distribution range of species in and around the Dadu Valley, and the dotted line represents the approximate distribution boundary of these species.The elevation data were obtained from Geospatial Data Cloud (2022).

Figure 2 .
Figure2.The phylogenetic tree of the genus Diploderma inferred from the mitochondrial ND2 (974 bp).ML bootstrap support/Bayesian posterior probability support is denoted above each node.The maximum parsimony strict consensus tree for the Diploderma species is inferred from ND2 gene fragments by using IQ-TREE v2.2.0[20]; the ND2 gene majority-rule consensus tree for the Diploderma species is inferred from partitioned Bayesian analyses by using MrBayes v3.2.7a[21].In view of the unique clades of the Yibin and Leibo populations, sp.1 and sp.2 are used instead, respectively.

Figure 2 .
Figure 2. The phylogenetic tree of the genus Diploderma inferred from the mitochondrial ND2 (974 bp).ML bootstrap support/Bayesian posterior probability support is denoted above each node.The maximum parsimony strict consensus tree for the Diploderma species is inferred from ND2 gene fragments by using IQ-TREE v2.2.0[20]; the ND2 gene majority-rule consensus tree for the Diploderma species is inferred from partitioned Bayesian analyses by using MrBayes v3.2.7a[21].In view of the unique clades of the Yibin and Leibo populations, sp.1 and sp.2 are used instead, respectively.

Animals 2024 ,
14, x FOR PEER REVIEW 13 of 22the vertebral line between the dorsolateral stripes from the neck to the base of the tail in both sexes.When stressed, the color of ventral head turns dim-gray (105, 105, 105) with slightly larger scales turning white (255, 255, 255), the ventral body coloration turning dim-gray, and the ventrolateral body turning black (0, 0, 0).

Table 2 .
Morphometric comparison between Diploderma daduense sp.nov, sp.1, sp.2, and D. splendidum.Morphometric measurements are in the unit of mm.For measurement and count methods and abbreviations, see Section 2.