Another new species of Dixonius (Squamata, Gekkonidae) from Gia Lai Province in the Central Highlands, Vietnam

Another new species of Dixonius , D. fulbrighti sp. nov. , is described from Gia Lai Province, in the Central Highlands of Vietnam, using an integrated approach based on morphological, categorical (color pattern), and mitochondrial NADH dehydrogenase sub-unit 2 gene (ND2) and its flanking tRNAs data. Phylogenetic analyses recovered Dixonius fulbrighti sp. nov. as closely related to D. gialaiensis from Gia Lai Province and part of a clade that includes D. minhlei , D. siamensis , and D. somchanhae . Multivariate (PCA, DAPC, and MFA) and univariate (ANOVA) analyses of 15 meristic (scale counts), six morphometric (mensural), and five categorical (color pattern and morphology) characters from 44 specimens of all eight species of Dixonius from Vietnam, Laos, and Cambodia clearly demonstrated that Dixonius fulbrighti sp. nov. is statistically different and diagnostically distinct from all closely related species of Dixonius . This new species discovery highlights the underestimated gecko diversity and the importance of continued fieldwork in the Central Highlands of Vietnam.


Introduction
The Central Highlands of Vietnam are composed of basaltic, quartzite, and granite substrates, and are mainly situated at elevations ranging from 800-2400 m, forming the central and southern parts of the Truong Son Mountain Range (Sterling et al. 2006). This upland mountain system is composed of "sky islands" that are isolated from each other by vast intervening lowlands, which have contributed to the evolution of unique and distinct forms (Bain and Hurley 2011). Unfortunately, these regions are undergoing habitat loss due to the conversion of natural forests into agricultural farmland (Luu et al. 2020; as observed by Vinh Quang Luu). Previous studies on gecko species have mainly been focused in protected areas (e.g., Nazarov et al. 2008;Jestrzemski et al. 2013;Do et al. 2021) but our recent research indicates that geckos recently described in the Central Highlands are also found outside the network of protected areas (Luu et al. 2017(Luu et al. , 2023. There is still much to be learned about the mechanisms that contribute to the high levels of diversity within this morphologically and ecologically diverse gecko lineage. In Vietnam, Dixonius species are distributed in the southern regions from Gia Lai to Dong Nai provinces, with seven known species, including D. aaronbaueri Ngo & Ziegler, 2009 from Ninh Thuan and Binh Thuan provinces; D. gialaiensis Luu, Nguyen, Le, Grismer, Ha, Sitthivong, Hoang & Grismer, 2023;D. melanostictus (Taylor, 1962) from Dong Nai Prvince (Nguyen et al. 2009); D. minhlei Ziegler, Botov, Nguyen, Bauer, Brennan, Ngo & Nguyen, 2016 from Dong Nai Province; D. siamensis (Boulenger, 1898) from Ninh Thuan, Gia Lai, Lam Dong,

Species delimitation
The general lineage concept (GLC: de Queiroz 2007) adopted herein proposes that a species constitutes a population of organisms evolving independently from other such populations owing to a lack of, or limited gene flow. By "independently," it is meant that new mutations arising in one species cannot spread readily into another species (Barraclough et al. 2003;de Queiroz 2007). Molecular phylogenies recovered multiple monophyletic mitochondrial lineages of individuals (populations) that were used to develop initial species-level hypotheses -the grouping stage of Hillis (2019). Discrete color pattern data and univariate and multivariate analyses of morphological data were then used to search for characters and morphospatial patterns consistent with the tree-designated species-level hypotheses -the construction of boundaries representing the hypothesis-testing step of Hillis (2019) -thus providing independent diagnoses to complement the molecular analyses. In this way, delimiting (phylogeny) and diagnosing (taxonomy) species are not conflated (Frost and Hillis 1990;Frost and Kluge 1994;Hillis 2019).
Genomic DNA was extracted from all liver tissues stored in ethanol following the standard protocols of DNeasy blood and tissue kit, Qiagen (California, USA). The PCR volume consisted of 20µl (1µl each primer, 7µl water, 10µl of Taq mastermix and 1 µl DNA template). PCR conditions were: 95 °C for 5min, followed by 42 cycles: 95 °C for 30s, 50 °C for 45s and 72 °C for 60s with a final elongation step for 6 min at 72 °C. PCR products were visualized using electrophoresis through a 1.2% agarose gel, marker 100 bp, 1X TAE and stained with Red-Safe Nucleic Acid Staining Solution and photographed under UV light of Geldoc system (Quantum CX5, Villber, France). Successful amplifications were purified using innuPREP Gel Extraction Kit (Analytik Jena, Germany). Cleaned PCR products were sent to Genewiz from Azenta Life Sciences for sequencing in both directions.
We obtained 1,431 base pairs from the NADH dehydrogenase subunit 2 gene (ND2) and the flanking tRNAs from 32 ingroup samples of Dixonius representing 14 nominal species, including the new samples from Gia Lai Province Vietnam. Heteronotia spelea was used as an outgroup to root the trees following Gamble et al. (2015). Sequence data for the other species were downloaded from GenBank. Newly generated sequences were deposited in GenBank (Table 1).
Maximum Likelihood (ML) and Bayesian Inference (BI) were used to estimate phylogenetic trees. Best-fit models of evolution determined in IQ-TREE  using the Bayesian information criterion (BIC) implemented in ModelFinder (Kalyaanamoorthy et al. 2017) indicated that F81+F was the best-fit model of evolution for the tRNAMET and K2P+I, and HKY+F+G4 were the best models of evolution for tRNAs2 and ND2, respectively. The ML analysis was performed using the IQ-TREE webserver (Trifinopoulos et al. 2016) with 1000 bootstrap pseudoreplicates using the ultrafast bootstrap (UFB) analysis (Minh et al. 2013;Hoang et al. 2018). The BI analysis was performed on CIPRES Science Gateway (Miller et al. 2010) using MrBayes v3.2.4 (Ronquist et al. 2012) using default models of evolution. Two independent runs were performed using Metropolis-coupled Markov Chain Monte Carlo (MCMCMC), each with four chains: three hot and one cold. The MCMCMC chains were run for 80,000,000 generations with the cold chain sampled every 8000 generations and the first 10% of each run being discarded as burn-in. The posterior distribution of the trees from each run were summarized using the sumt function in MrBayes v3.2.4 (Ronquist et al. 2012). Stationarity was checked in Tracer v1.6 (Rambaut et al. 2014) to ensure effective sample sizes (ESS) for all parameters were well above 200. We considered Bayesian posterior probabilities (BPP) of 0.95 and above and ultrafast bootstrap support values (UFB) of 95 and above as an indication of strong nodal support (Huelsenbeck et al. 2001;Minh et al. 2013). Uncorrected pairwise sequence divergences (p-distance) were calculated in MEGA 11 (Kumar et al. 2016) using the complete deletion option to remove gaps and missing data from the alignment. A time-calibrated Bayesian phylogenetic tree was estimated using BEAST 2 (Bayesian Evolutionary Analysis by Sampling Trees) version 2.7.3 (Drummond et al. 2012) implemented in CIPRES (Cyberinfrastructure for Phylogenetic Research; Miller et al. 2010) where the ingroup node subtending the split between Dixonius aaronbaueri and the remaining species was given a 24.04 mya prior with an offset range of 20.23-27.68 mya following Gamble et al. (2015). The split between Heteronotia and Dixonius was dataed at 45.0 mya with an offset range of 33.3-56.8 mya (Gamble et al. 2015). An input file was constructed in BEAUti (Bayesian Evolutionary Analysis Utility) version 2.7.3. An optimised relaxed clock with unlinked site models, linked clock and tree models, using a Calibrated Yule prior were employed for the species level analysis. BEAST Model Test (Bouckaert and Drummond 2017), implemented in BEAST, was used to numerically integrate over the uncertainty of substitution models while simultaneously estimating phylogeny using Markov chain Monte Carlo (MCMC). MCMC chains were run for 60 million generations and logged every 6,000 generations. The BEAST log file was visualized in Tracer v. 1.7.2 (Rambaut et al. 2014) to ensure effective sample sizes (ESS) were above 200 for all parameters. A maximum clade credibility tree using mean heights at the nodes was generated using TreeAnnotator v.2.7.3 (Rambaut and Drummond 2013) with a burnin of the first 10% of each run. Nodes with Bayesian posterior probabilities (BPP) of 0.95 and above were considered strongly supported (Hulsenbeck et al. 2001;Wilcox et al. 2002).

Morphological data and analysis
The morphological data set comprised four individuals of the new Dixonius population from Duc Co District, Gia Lai Provine, Vietnam (VNUF R.2022.81-84) and seven closely related species based on the phylogeny (see below), including three type specimens of D. gialaiensis from Gia Lai Province, Vietnam (VNUF R.2020(VNUF R. .22, 2020(VNUF R. .33, 2020 1, 2016.3, 2016.4, VNMN R.2016. Morphometric and meristic data were taken from the 44 specimens following Bauer et al. (2004) and Ngo and Ziegler (2009). Morphometric characters were measured after preservation with a digital caliper to the nearest 0.1mm and a zoom stereomicro scope on the right/left of the body and included SVL: snout-vent length (taken from the tip of the snout to the vent), TaL: tail length (taken from the vent to the tip of the tail, original or partially regenerated), TW: tail width (taken at the base of the tail immediately posterior to the postcloacal swelling), BW: body width (greatest width of torso, taken at the level of midbody), HL: head length (the distance from the posterior margin of the retroarticular process of the lower jaw to the tip of the snout), HW: head width (measured at the angle of the jaws), HD: head depth (the maximum height of head measured from the occiput to base of the lower jaw), EL: ear length (greatest oblique length across the auditory meatus), TBL: Tibia length (taken on the ventral surface from the posterior surface of the knee while flexed 90° to the base of the heel), AG: axilla to groin length (taken from the posterior margin of the forelimb at its insertion point on the body to the anterior margin of the hind limb at its insertion point on the body), FA: forearm length (taken on the ventral surface from the posterior margin of the elbow while flexed 90° to the inflection of the flexed wrist), ED: eye diameter (the greatest horizontal diameter of the eye-ball), EN: eye nostril distance (measured from the anterior margin of the bony orbit to the posterior margin of the external nares), ES: eye snout distance (measured from anteriormost margin of the bony orbit to the tip of snout), EE: eye ear distance (measured from the anterior edge of the ear opening to the posterior margin of the eye-ball), IN: internarial distance (measured between the external nares across the rostrum), IO: interorbital distance (measured between the dorsal-most edges of the bony orbits).
Meristic characters included V: ventral scales (counted transversely across the abdomen midway between limb insertions from one ventrolateral fold to the other), DTR: longitudinal rows of dorsal tubercles (counted transversely across the body midway between the limb insertions from one ventrolateral body fold to the other), PV: paravertebral scales (counted in a paravertebral row from first scale posterior to parietal scale to last scale at the level of vent opening), PV': paravertebral scales (counted in a row between limb insertions), T4: lamellae under fourth toe (from the distal scale containing claw to basal scale that broadly contacts adjacent fragmented scales), IOS: Interorbital scales (counted at narrowest point between orbits across the frontal bone), ICS: interciliary scales (counted between supraciliaries at midpoint of orbit), SPL: supralabials (counted from the largest scale at the corner of the mouth to the rostral scale), IFL: infralabials (counted from termination of enlarged scales at the corner of the mouth to the mental scale), MO: number of supralabial at midorbital position, PP: number of precloacal pores in males.
Color pattern characters on dorsum included the presence or absence of canthal stripes (CanthStrp), presence or absence of strong darkly barred lips (LipBar), presence or absence of dark-colored round blotches on the top of the head (RdHdBlch) and dorsum (RdBodBlch), and presence or absence of two regularly arranged rows of whitish tubercles on flanks (Tub). The raw morphological data for all characters and specimens are presented in Tables 2, 3.  (Thorpe 1975(Thorpe , 1983Turan 1999;Lleonart et al. 2000, accessible in the R package GroupStruct (available at https://github.com/chankinonn/GroupStruct). The morphometrics of each species were normalized separately and then concatenated into a single data set so as not to conflate potential intra-with interspecific variation (Reist 1986;McCoy et al. 2006). All data were scaled to their standard deviation to ensure they were analyzed on the basis of correlation and not covariance. Meristic characters (scale counts) used in the statistical analyses were SPLr/l, IFLr/l, MO, IOS, ICS, V, DTR, and T4r/l. Paried meristics were averaged if they differed. Precloacal and femoral pores were omitted from the multivariate analyses due to their absence in females. Categorical color pattern characters analyzed were CanthStrp, LipBar, Rd-HdBlch, RdBodBlch, and Tub. Analyses of variance (ANOVA) were conducted on meristic and normalized morphometric characters (see below) with statistically similar variances -following a Levene's test; p > 0.05 -to search for the presence of statistically significant mean differences (p < 0.05) among species across the data set. Following the ANOVAs, each data set was subjected to a TukeyHSD test to ascertain which species pairs differed significantly from each other for which particular characters. Boxplots were generated for discrete meristic characters in order to visualize the range, mean, median, and degree of differences between pairs of species bearing statistically different mean values and violin plots were generated for continuous morphometric characters to visualize the same.
Morphospatial positions were visualized using principal component analysis (PCA) from the ADEGENET package in R (Jombart et al. 2010) on the normalized and meristic data to determine if lineage positioning was consistent with the putative species boundaries delimited by the molecular phylogenetic analyses and defined by the univariate analyses (see above). PCA, implemented using the "prcomp()" command in R, is an unsupervised analysis plotting the overall variation among individuals (i.e. data points) while treating each individual independently (i.e. not coercing data points into pre-defined groups). Subsequent to the PCA, a discriminant analysis of principle components (DAPC) was used to test for corroboration and further discrimination of morphospatial differences among the putative species. DAPC a priori groups the individuals of each predefined population inferred from the phylogeny into separate clusters (i.e., plots of points) bearing the smallest within-group variance that produce linear combinations of centroids having the greatest between-group variance (i.e. linear distance; Jombart et al. 2010). DAPC relies on standardized data from its own PCA as a prior step to ensure that variables analyzed are not correlated and number fewer than the sample size. Principal components with eigenvalues accounting for 90-95% of the variation in the data set were retained for the DAPC analysis according to the criterion of Jombart et al. (2010).
To test and further corroborate the PCA and DAPC analyses, we conducted a multiple factor analysis (MFA) on the above mentioned morphological characters plus the categorical color pattern characters for a near total evidence data set (see Tables 5, 6). The MFA was implemented using the mfa() command in the R package FactorMineR (Husson et al. 2017) and visualized using the Factoextra package (Kassambara and Mundt 2017). MFA is a global, unsupervised, multivariate analysis that incorporates qualitative and quantitative data (Pagès 2015), making it possible to analyze different data types simultaneously in a nearly total evidence environment. In a MFA, each individual is described by a different set of variables (i.e. characters) which are structured into different data groups in a global data frame -in this case, quantitative data (i.e. meristics and normalized morphometrics) and categorical data (i.e. color pattern). In the first phase of the analysis, separate multivariate analyses are carried out for each set of variables -principal component analyses (PCA) for the quantitative data sets and multiple correspondence analysis (MCA) for categorical data. The data sets are then normalized separately by dividing all their elements by the square root of their first eigenvalues. For the second phase of the analysis, the normalized data sets are concatenated into a single matrix for a global PCA of the data. Standardizing the data in this manner prevents one data type from overleveraging another. In other words, the normalization of the data in the first phase prevents data types with the most number of characters or the greatest amount of variation from outweighing other data types in the second phase. This way, the contribution of each data type to the overall variation in the data set is scaled to define the morphospatial distance between individuals as well as calculating each data type's and each character's contribution to the overall variation in the data set (Pagès 2015;Kassambara and Mundt 2017).

Molecular results
The ML, BI, and BEAST analyses yielded trees with identical topologies and strong support at almost every node (see Fig. 2). The molecular analyses revealed that Dixonius aaronbaueri is the sister species to a clade containing all other Dixonius species. Additionally, all analyses supported the newly discovered population from Duc Co District, Gia Lai Province, Vietnam, as the well-supported sister species (1.00/100) of the clade containing D. minhlei and D. gialaiensis (see Figs 2, 3). The uncorrected pairwise sequence divergences among Dixonius species ranged from 2.57% to 18.84% (see Table 4). The new population from Duc Co District, Gia Lai Province, had a range of 3.12% to 16.43% and is most similar to D. gialaiensis and most distant from D. aaronbaueri.

Statistical analyses
The first two principal components (PC1 and PC2) of the PCA analysis recovered 56.6% of the variation in the morphometric and meristic data set (see Fig. 4A). These components loaded most heavily for body width (BW), head width (HW), eye nostril distance (EN), eye snout distance (ES), and eye ear distance (EE) along PC1, and interorbital distance (IO), supralabials (SPLr/l), number of supralabial at midorbital position (MO), and ventral scales      Table 6. Summary statistics of normalized morphometric and meristic characters among the Dixonius species. (V) along PC2 (Table 5). The PCA analysis indicates that the population from Duc Co District, Gia Lai Province is widely separated from all other species along the ordination of the first two axes except for the distantly related D. vietnamensis. Moreover, the new species is well-separated from all other species in the DAPC except for slight overlap with D. minhlei in their 67% inertia ellipses (Fig. 4B).
The MFA analysis recovered all species being separated from each other, including the population from Duc Co District, Gia Lai Province and its close relative D. gialaiensis and D. minhlei (Fig. 5A). The new population plots closest to the distantly related D. vietnamensis. The morphometric data contributed to approximately 40% of the 31.6% of the variation along Dim-1, followed by the categorical and meristic data. For Dim-2, the categorical data contributed 80% of the 17.8% of the variation, followed by morphometric and meristic data. For Dim-3, the meristic data contributed 70% of the 12.0% of the variation, followed by morphometric and categorical data. For Dim-4, the meristic data contributed 50% of the 8.5% of the variation, followed by categorical and morphometric data (Fig. 5B).
The ANOVAs and subsequent TukeyHDS tests demonstrated that the population from Duc Co District, Gia Lai Province bears statistically different mean values from all other species in various combinations of characters (Tables 6, 7) and differs significantly from the closely related D. gialaiensis, in head length (HL), head width (HW), head depth (HD), eye diameter (ED), and tibia length (TBL) (Fig. 7; Tables 6, 7). Given that we had only one specimen of the closely related D. minhlei we could not conduct any statistical analyses including it. However, the two do differ discretely in head length (HL), body width (BW), head depth (HD), eye diameter (ED), eye nostril distance (EN), eye snout distance (ES), forearm length (FA), tibia length (TBL), and axilla to groin length (AG) as well as in color pattern of two rows of regularly disposed whitish tubercles on the dorsal surface in the Duc Co District, Gia Lai Province population as opposed to their absence in D. minhlei. Variation in all metric characters is visualized in Figs 6, 7.
Given the phylogenetic placement of the population from Duc Co District, Gia Lai Province not being embedded within any other species, its statistically different mean values in a number of morphometric and meristic characters among the other species, and its generally isolated morphospatial placement in all three morphometric analyses, we consider this population to be an evolutionarily unique and statistically diagnosable lineage and therefore describe it below as a new species.  Diagnosis. Dixonius fulbrighti sp. nov. can be separated from all other species of Dixonius by possessing the unique combination of having a maximum SVL of 46.0 mm; 16-20 longitudinal rows of dorsal tubercles at midbody; 22-24 longitudinal rows of ventrals across the abdomen; sevennine supralabials, sixth in at midorbital position; five-seven infralabials; eight or nine interorbital scales; seven precloacal pores in the adult male, femoral pores lacking; seven precloacal-pitted scales, femoral pores absent in adult female; 13-15 lamellae on fourth toe; dorsal ground color grey-brown with the presence of thick, irregular-shaped, black brown blotches from head to body; canthal stripe extending from the nostrils continuing behind orbit to back of head; dark bars on the lips absent; two rows of regularly disposed whitish tubercles along the flanks to originale portion of tail. These characters are scored across all Dixonius species from Vietnam and Laos in Tables 6 and 7.
Description of the holotype. Adult male, SVL 46.0 mm; head moderate in length (HL/SVL 0.30), wide (HW/HL 0.57), depressed (HD/HL 0.42), distinct from neck; prefrontal region concave; canthus rostralis rounded; snout elongate (ES/HL 0.35), rounded in dorsal profile; eye moderate size (ED/HL 0.16); ear opening oval, obliquely oriented, moderate in size; diameter of eye smaller than eye to ear distance (ED/EE 0.59); rostral rectangular, partially divided dorsally by straight rostral groove, bordered posteriorly by large left and right supranasals, bordered laterally by first supralabials; external nares bordered anteriorly by rostral, dorsally by large supranasal, posteriorly by two smaller postnasals, bordered ventrally by first supralabial; 8,8 (R,L) rectangular supralabials extending to below and slightly past posterior margin of eye, sixth in midorbital position; 6,7 (R,L), infralabials tapering smoothly to just below midpoint of eye, decreasing gradually in size; scales of rostrum and lores flat to domed, larger than granular scales on top of  head and occiput; scales of occiput intermixed with distinct, small, conical tubercles; superciliaries elongate, largest anteriorly; mental triangular, bordered laterally by first infralabials and posteriorly by two postmentals (large right trapezoidal shape and left irregular shape) contacting medially for 60% of their length posterior to mental; gular and throat scales small, granular, grading anteriorly into slightly larger, flatter, smooth, imbricate, pectoral and ventral scales.
Body relatively short (AG/SVL 0.44); dorsal scales small, granular interspersed with larger, conical, regularly arranged, keeled tubercles; tubercles extend from top of head onto posterior half of original tail forming longitudinal rows, terminating at last portion of orginale tail; smaller tubercles extend anteriorly onto nape and occiput, diminishing in size and distinction on top of head; 18 longitudinal rows of tubercles at midbody; 32 paravertebral scales, number of scales in a paravertebral row from first scale posterior to parietal scale to last scale at the level of vent opening; 22 paravertebral scales in a row between limb insertions; 24 flat, imbricate, ventral scales much larger than dorsal scales; 7 enlarge, pore-bearing, precloacal scales in an angular series; and no deep precloacal groove or depression.
Forelimbs moderate in stature, relatively short (FA/SVL 0.12); granular scales of forearm slightly larger than those on body, interspersed with small tubercles; hind limbs more robust than forelimbs, moderate in length (TBL/SVL 0.15), covered dorsally by granular scales interspersed with large, and small conical tubercles; ventral scales of thigh flat, imbricate, larger than dorsals; subtibial scales flat, imbricate; proximal femoral scales smaller than distal femorals; femoral pores absent; digits relatively long with 14 lamellae on fourth toe; and claws well developed.
Regenerated tail 46.4 mm in length, 5.4 mm in width at base, tapering to a point; dorsal scales of tail flat, oval with conical, keeled tubercles in anterior part; median row of transversely expanded subcaudal scales, significantly larger than dorsal caudal scales on original portion; base of tail bearing hemipenal swellings; and postcloacal scales flat, imbricate.
Coloration in life. (Fig. 8) Dorsal ground color of grey-brown with thick, irregular-shaped, black to brown blotches extending from head to the body; canthal stripe extending from behind the nostrils conitinuing behind orbit to back of head; dark bars on lips; uneven light-colored spots running from postorbital region along the flanks to the base of tail; dorsal surface of fore-and hind limbs uniformly light-grey with dark-brown spots; dorsum of orginal tail covered with some small black to brown blotches; all ventral surfaces uniformly beige.
Variation. (Fig. 8) The female paratypes (VNUF R.2022.82-84) are generally uniformly brown dorsally with uneven light-colored spots running from the postorbital region along the flanks to the tail tip; dorsum of posterior part of tail grey with narrowed light-colored bands. Additional measurements are in Tables 2, 3, 8. Distribution. Dixonius gialaiensis sp. nov. is currently known only from the type locality of in Grong Village, Ia Krieeng Commune, Duc Co District, Gia Lai Province, Central Highlands, Vietnam (Fig. 1).
Natural history. The specimens were found on the forest floor, during the evening between 1900 hrs and 2000 hrs. The surrounding habitat consisted of secondary forest with Narra Padauk (Pterocarpus macrocarpus) trees as the dominant species and shrub trees on the forest floor (Fig. 9). Currently, the area is managed by the local commune, but it is not part of any protected area system. Therefore, further investigation on the population status is needed to suggest appropriate conservation measures.
Etymology. The new species is named after Mr. J. William Fulbright, the founder of the Fulbright Program which has provided opportunities for thousands of students, scholars, and professionals from around the world to study, teach, and conduct research in the United States and other countries in order to promote a greater understanding and cooperation between nations.

Discussion
Dixonius fulbrighti sp. nov. is most closely related to the sister species D. minhlei and D. gialaiensis, but can be distinguished from the both species by differences in body shape and color pattern (see Table 5). The molecular analysis revealed a 3.12-3.61% uncorrected pairwise genetic distance between Dixonius fulbrighti sp. nov. and D. gialaiensis and D. minhlei, and all evolved separately in geographically isolated regions. The type locality of D. fulbrighti sp. nov. is approximately 55 km west of the type locality of D. gialaiensis, and the two areas are separated by fragmented habitats bearing residential areas and transport systems, including the National Road AH17, and the Local Roads DT663 and DT6754 (Fig. 1). Moreover, the type locality of Dixonius fulbrighti sp. nov. is approximately 280 km northeast of the type locality of D. minhlei, and these two localities are isolated by different habitat types and river systems. Additionally, the divergence time estimated by the BEAST analysis indicates that Dixonius fulbrighti sp. nov. and the D. minhlei -D. gialaiensis clade have been evolving independently for a considerable period of time, approximately 4.12 million years ago. This is likely due to their historical isolation from each other in geographically distinct regions, that is maintained by current barriers to gene flow as well as by human activities such as road construction and deforestation, helping to maintain their separation. The fragmented habitats between the type localities of the these species suggest these barriers are continuing to impact the distribution and evolution of geckos in this region (Grismer et al. 2013;Epps and Keyghobadi 2015). Further research on the biogeography and genetic diversity of these species will be important for understanding their evolutionary history and informing conservation efforts to protect their unique habitats.