Nghiên cứu đa dạng thành phần loài và quan hệ di truyền của họ Tắc kè (Gekkonidae) ở một số khu vực núi đá vôi tại nước Cộng hòa Dân chủ Nhân dân Lào tt

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM NATIONAL UNIVERSITY OF FORESTRY SALY SITTHIVONG SPECIES DIVERSITY AND PHYLOGENETIC RELATIONSHIPS OF THE FAMILY GEKKONIDAE IN LIMESTONE AREAS FROM LAO PEOPLE’S DEMOCRATIC REPUBLIC MAJOR: FOREST MANAGEMENT CODE: 62 02 11 SUMMARY OF THE DOCTORAL DISSERTATION HANOI, 2022 The thesis was completed at the Vietnam National University of Forestry Supervisor: Assoc Prof Dr Luu Quang Vinh – Vietnam National University of Forestry Prof Dr Nguyen The Nha – Vietnam National University of Forestry Reviewer 1: Reviewer 2: Reviewer 3: The dissertation will be defended at the Doctoral Evaluation Dissertation Council: - Date and time: - Place: Vietnam National University of Forestry The dissertation can be found at: - National Library of Vietnam - Library of Vietnam National University of Forestry INTRODUCTION Justification for the research Knowledge on the composition of reptiles in the world during a recent decade has increased significantly from 9,300 species in the begining of 2011 to 11,570 species in 2021 (Uetz et al 2021) However, according to Boehm et al (2013), an estimated 20% of reptiles globally are threatened with extinction Besides, the conservation of reptiles also faces many difficulties due to the limited understanding of their diversity and bio-ecological characteristics Lao People's Democratic Republic (hereafter Laos) is one of the countries with a high diversity of flora and fauna, due to its geographical location, climate and mountainous terrain accounts for most of the area compared to other countries However, studies on reptiles (Reptilia) are limited, especially the Gecko family (Gekkonidae) In recent years, species discovery studies in the family Gekkonidae are being known with many species discovered and described in Laos, and these findings are mainly concentrated in the limestone karst ecosystem However, the number of known species is still underestimated in comparision with these species from neibouring countries such as Vietnam and Thailand, creating the question that how many species of geckos are in Laos? The limestone ecosystem is considered an ideal natural laboratory for the study of taxonomy, ecology, evolution and zoology The limestone mountain contains many different habitats and caves, so the fauna is often highly endemic In Laos, limestone mountains are mostly distributed in the central region and some northern provinces (Viossanges, 2017) As a result, this thesis focuses on karst areas of the central and northern provinces of Laos where have been poorly studied For mentioned reasons, I carried out the thesis “Species diversity and phylogenetic relationships of the family Gekkonidae in limestone areas from Lao People’s Democratic Republic” The study’s results have been provided a raw evidence on the diversity of species composition, taxonomy and genetic relationships of some genera of the Gecko family, information on the distribution characteristics of species in the Gecko family In addtion, the similarity relationship in species composition among the study areas and propose solutions for Gecko conservation in Laos have been evaluated Goal and objectives 2.1 Goal Provide scientific data on the species diversity and distribution characteristics, in order to propose solutions for the Gecko family (Gekkonidae) conservation in the limestone areas of Laos 2.2 Objectives - To determine the species diversity level of the family Gekkonidae in the study area - To identify species composition and genetic relationships of species Cyrtodactylus, Dixonius and Gekko - To evaluate the similarity in the composition of gecko species among the study sites - To assess the current status of Gecko species distribution according to habitat, altitude and location - To identify threat factors and conservation status of gecko species in Laos - To propose solutions to conserve the family Gekkonidae in the limestone ecosystem of Laos Subject and scope - Research subject: Species belonging to Gekkonidae - Scope: Limestone areas in some provinces of Northern and Central Laos namely: Vientiane, Luangprabang, Khammuane, Udomxay , Huaphane and Xiengkhoang provinces Contributions - 28 gecko species have been recorded in the study areas - Specific morphological characteristics for 28 collected species have been described with specific data on Gekko species distribution in the study areas as well as genetic relationships of the genera in the Gecko family - Species composition similarity and distribution characteristics according to habitat, altitude and location have been evaluated - Current status, threats and proposing solutions for gecko conservation in the limestone mountains of Laos have been assessed Scientific and practical significance 5.1 Scientific significance - Described new species to science and recorded new provincial records of geckos from Udomxay Province - Updated information on species composition, distribution characteristics of geckos in forest areas on limestone mountains in Northern and Central Laos: Vientiane, Luangprabang, Khammuone, Udomxay, Huaphane and Xiengkhoang provinces - Provided information on morphological characteristics and evaluated genetic relationships of the genera Cyrtodactylus, Dixonius and Gekko 5.2 Practical significance The dissertation has provided information as a scientific basis for the planning and management of biodiversity conservation in Northern and Central Laos depending on: 1) Confirming prioritized areas for conservation 2) Identifying prioritized species for conservation 3) Establishing crucial activities for gecko species conservation Thesis outline The dissertation consists of 142 pages, including: Introduction pages; Chapter 1: Overview 14 pages; Chapter 2: Contents, methods and study sites 18 pages; Chapter 3: Results and discussion 67 pages; Conclusions, existences and recommendations pages; References 10 pages Moreover, there are 15 tables and 65 figures in this dissertation Chapter Overview The dissertation has overviewed and summarized about main relevant issues in the world, in Vietnam and Laos: (1) Overview of reptiles research in Laos; (2) Taxonomy and distribution system of the Gecko family (Gekkonidae); (3) Other related studies Overview of reptiles research in Laos The overview of the research problems has helped to have a correct and comprehensive understanding of the situation of reptile research in Laos Accordingly, the total number of recorded and described reptile species in Laos is 181 species in February 2018 (Uetz et al 2018), there is an increasing number of total reptile species from 212 species in 2020 to 225 species in May 2021 Taxonomy and distribution system of the Gecko family (Gekkonidae) A research overview provided how to identify the taxonomic features and distribution of Gekkonidae The outstanding taxonomic features can be summarized as: Gekkonidae (Animalia), Vertebrata (Chordata), Reptilia and Squamata They live mainly in warm climates around the world Geckos (Gekkonidae) have unique vocalizations among lizards, they use sound to communicate with each other Most geckos not have eyelids but have transparent membranes, which are cleaned by licking The majority of gekko emit not only foul odors but also droppings on their predators in self-defense Besides, many species have suckers under their toes that allow them to cling to tree trunks, ceilings and walls easily In terms of species diversity, Laos has 45 gecko species belonging to genera in 2018, a described species in 2010 as Cnemaspis laoensis; the genus Cyrtodactylus is the genus with the largest number of species in Laos In addition, there are 58 genera of Gekkonidae with 1,430 species in the world updated in May 2021 Other related studies Previous studies only selected a few representative species but did not include all species of Gekkonidae as for species diversity and genetic relationships in the limestone areas of Laos In this study is to investigate and collect specimens in limestone mountains in Northern and Central Laos, in order to gather available data serving to Gecko family (Gekkonidae) diversity and genetic relationships Chapter 2: CONTENTS, METHODS AND STUDY SITES 2.1 Time and place of the study The field surveys have been conducted from June 2018 to April 2020 in a total of 112 days for field survey times, 13 sites, 38 survey transects Survey efforts are shown in Table 2.1 Table 2.1: Time and place for field trips Stage Location Vangvieng District Phuang District Namor District Xay District Ngoi District Luangprabang District Phu Kut District Nonghet District Khunham District Bualapha District Hiem District Xon District Viengxay District Coordinates 18°54’44” N; 102°27’05” E 18°39’21” N; 102°06’55” E 20°54’43” N; 101°45’37” E 20°41’05” N; 101°59’21” E 20°42’33” N;102°40’32” E 19°52’32” N; 102°08’36” E 19°34’25” N’;103°04’55” E 19°29’52” N; 103°59’05” E 18°12’25” N;104°31’36” E 17°29’03” N; 105°35’24” E 20°04’59” N; 103°22’10” E 20°27’25” N;103°20’57” E 20°25’08” N;104°13’49” E Altitude (m) 230-684 232-675 620-827 642-782 348-702 298-439 1.080-1.205 1.105-1.397 168-524 170-214 687-1.270 715-926 698-985 Time 25/7-9/8/2018 23/10-6/11/2018 21/4-28/4/2019 29/4-6/5/2019 7/5-13/5/2019 14/5-20/5/2019 10/7-16/7/2019 17/7-23/7/2019 26/10-2/11/2019 25/2-3/3/2020 10/3-16/3/2020 17/3-23/3/2020 26/3-2/4/2020 2.2 Research contents - Investigation of species diversity of Gekkonidae in limestone mountain areas of Northern and Central Laos: karst mountains and caves in Vientiane, Luangprabang, Khammuone, Udomxay, Huaphane and Xiengkhoang - Determination of species composition and evaluation of genetic relationships between species and populations in the Bent-toed Gecko (Cyrtodactylus), Leaf-toed Gecko (Dixonius) and Gecko (Gekko) recorded in Laos - Comparison of the similarity in species composition of the gecko family among the study sites and between the North and the Central region - Evaluation of the species distribution characteristics in the Gecko family by altitude ranges, habitat types, by micro-habitat (location: on trees, on the ground, cliffs) - Assessment of threat factors to populations of Gekkonidae species at the study sites and solutions for conservation 2.3 Materials and Methods 2.3.1 Field survey Point and transect surveys have been applied in this study To be more specific, the transect surveys were made based on the topographic map, vegetation and habitat of the Gecko species These transects were designed through different habitat types and elevations of the study area, paying special attention to the limestone mountains with caves and cliffs, the valleys between the limestone mountain ranges in the forest Each transect survey has be recorded by GPS using trackmaker Survey time was at night from 17h00 to 23h00 2.3.2 Research materials 1) Chemicals The chemicals used to separate the total DNA including: Dneasy Blood Kit and Tissue (Qiagen, Germany); GenJet Genomic DNA Purification (ThermoFisher Scientific, Lithuania); ethanol (Merck, Germany) PCR reactions were performed using a mixture of HotStar Taq mastermix (Qiagen, Germany) and DreamTaq Mastermix (ThermoFisher Scientific, Lithuania) PCR products were visualized by electrophoresis using the following reagents: agarose, ethidium bromide, tris base, EDTA, marker kb, marker 100 bp (1st Base, Malaysia) and dye 6x (ThermoFisher Scientific, Lithunia) Successful PCR products were purified using the GeneJET PCR Purification Kit (ThermoFisher Scientific, Lithuania) 2) Primers for PCR reactions Primers used in the genetic relationship study of the Bent-toed Gecko (Cyrtodactylus), the Leaf-toed Gecko (Dixonius) and the Gecko (Gekko) were referenced to the previous study of Ivanova et al 2006, Macey et al 1997 and Greenbaum et al 2007 The catalyst process was shown in tables 2.3-2.5 Table 2.3 Primers used in studying genetic relationships of Bent-toed Gecko (Cyrtodactylus) Primer name Primer sequences Source VF1-d 5’- TTCTCAACCAACCACAARGAYATYGG -3’ Ivanova et al 2006 VR1-d 5’- TAGACTTCTGGGTGGCCRAARAAYCA -3’ Ivanova et al 2006 Table 2.4 Primers used in studying genetic relationships of Leaf-toed Gecko (Dixonius) Primer name Primer sequences Source MetF1 5’- AAGCTTTCGGGCCCATACC -3’ Macey et al 1997 COIR1 5’- AGRGTGCCAATGTCTTTGTGRTT -3’ Macey et al 1997 Table 2.5 Primers used in studying genetic relationships of Gekko genus (Gekko) Primer name Primer sequences Source GF1 GR1 5'- CAAGCACHATYATYACYATAT -3' 5'-CCTATGTGTGCGATTGATGA-3’ Greenbaum et al 2007 Greenbaum et al 2007 3) Bioinformatics softwares The bioinformatics software used in the study included: Sequencher v5.4.6 (Gene Codes Corp., AnnArbor, MI, USA), ClustalX v2.1 (Thompson et al., 1997), jModeltest v2.1.4 (Darriba et al., 2012), Modeltest v3.7 (Posada and Crandal, 1998), MrBayes v3.2 (Ronquist et al., 2012), Tracer v1.5 (Rambaut and Drummond, 2009), Figtree v1.3 (Rambaut, 2009), PAUP v4.0b10 (Swofford, 2001) 4) Lab equipment Research equipment for genetic analyses belong to the Laboratory of Genetics Department, Ha Noi University of Natural Sciences at Vietnam National University; Wildlife Department in Faculty of Forest Resources and Environmental Management at Vietnam National University of Forestry 5) Field equipment Equipment for field investigation: GPS, digital caliperto the nearest 0.1 mm, monitoring sheet, thermohygrometer, digital camera, headlight, measuring tape, etiket, lighter, marker, alcohol, tongs, DNA sample tube, gloves 2.3.3 Research specimens Sampling method: Samples are collected by hand and specialized tools such as rubbercoated clamps are used to avoid harming the animals After measuring, photographing, collecting DNA samples, presentative samples will be kept as research specimens Specimen making: Specimens were anaesthetized and euthanized in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons, 2002) and fixed in approximately 85% ethanol, then later transferred to 70% ethanol for permanent storage Sampling for DNA analyses: Muscle, liver or tail tissue samples were collected and stored separately in 70% alcohol (Merk, Germany) at Wildlife Department, Faculty of Forest Resources and Environmental management at Vietnam National Umiversity of Forestry 2.3.4 Analysis of morphological characteristics and Gecko identification After collection, specimens were brought to the laboratory of the Vietnam National University of Forestry for analysis The morphological measurements were taken with an Etopoo digital caliper to the nearest 0.1 mm Scale counts were taken using a stereo microscope (Olympus SZ61) Measurements and scale counts followed Nguyen (2013), Luu (2015), and Ziegler (2016) Table 2.6 List of abbreviations and explanations of measurement and scale count characters Abbreviation Explanation Measurements (mm) SVL TaL TrunkL/AG HL HW HH SE EyeEar ForeL 10 FemurL 11 CrusL 12 LD4A 13 LD4P 14 OD 15 EarL 16 RW 17 RH 18 MW 19 ML Scale counts (1, 2, 3, ) 20 CS 21 N 22 I/IN 23 SL 24 IL 25 IO Snout-vent length Tail length Trunk length Head length Maximum head width Maximum head height Snout to orbit distance Orbit to ear distance Forearm length Femur length Crus length Length of finger IV Length of toe IV Greatest diameter of orbit Ear length Maximum rostral width Maximum rostral height Maximum mental width Maximum mental length Ciliary spines Nasals (nasorostrals,supranasals, postnasals) Intersupranasals Supralabials Infralabials Interorbitals Abbreviation 26 27 28 29 30 31 32 33 34 35 36 37 38 39 PO PM GP DTR GSDT SBL SR V LF1 LF4 LT1 LT4 PP PAT Explanation Preorbitals Postmentals Gulars bordering the postmentals Dorsal tubercle rows at midbody Granular scales surrounding dorsal tubercles Number of scales along the midbody Number of scale rows around midbody Ventral scales in longitudinal rows at midbody Subdigital lamellae under whole first finger Subdigital lamellae under whole fourth finger Subdigital lamellae under whole first toe Subdigital lamellae under whole fourth toe Precloacal pores Postcloacal tubercles After analyzing morphological data, detailed identifications of gecko specimens were followed Smith (1935), Taylor (1963), Dao (1979), Nguyen et al (2010, 2011), Hartmann et al (2013), Ziegler et al (2013, 2016), Nguyen et al (2014), Luu et al (2014, 2015, 2016), Vassilieva et al (2016), Schneider et al (2020) and other documents Furthermore, when the process of classifying samples finished, specimens were subsequently deposited in the collections of the Vietnam National University of Forestry (VNUF), the National University of Laos (NUOL), the Institute of Ecology and Biological Resources (IEBR), and the Zoologisches Forschungsmuseum Alexander Koenig, Bonn, Germany (ZFMK)., Systematics and nomenclature followed Nguyen et al (2009) and Uetz et al (2011) and some recently published documents are main references for the classification common names of taxonomic levels Figure 2.3 Measuring and counting indicators for gecko specimens 2.3.5 DNA extraction and sequencing Tissue samples were stored at 4°C prior to extraction The tissue used for extraction is taken from deep within the specimen mass to limit the risk of contamination The Dneasy Blood and Tissue Kit (Qiagen, Germany) is used for low-volume or long-collected samples and stored under uncertain temperature, solution conditions and GeneJet Genomic DNA Purification (ThermoFisher Scientific, Lithuania) for newly collected tissue samples, large amount of samples stored in guaranteed conditions (Merck alcohol 70%, Germany) The extraction was performed according to the manufacturer's instructions, with process-based adjustments The extraction process is carried out according to the following specific steps: sample pretreatment (cut the sample into small pieces, allow to dry and put in 1.5 ml eppendorf tube); membrane disruption and protein removal (using buffer ATL, AL-Qiagen, Germany; Digestion Solution, Lysis solution-ThermoFisher Scientific, Lithuania protein K-Qiagen, Germany); DNA precipitation (using Merck alcohol 50-100%, Germany); separation of DNA from other cell components (filter column containing a silica membrane); DNA purification (using buffer AW1, AW2-Qiagen, Germany; Wash 1, Wash 2ThermoFisher, Lithuania); DNA dissolution (buffer AE-Qiagen, Germany; Elution BufferThermoFisher Scientific, Lithuania) After extraction of total DNA, the obtained total DNA concentration was checked by electrophoresis on 1% agarose gel, 1X TBE buffer (Tris base, Boric acid, EDTA pH 8) at 70V for 30 Total DNA was compared with a kb marker and then visualized by ultraviolet light on an Alphamager MINI (Protein Simple, USA) PCR reaction was performed to diffuse the COI gene fragment of the mitochondrial genome with primer pairs In this study, with purpose for the next evolution research, the priority is to use the mastermix finished-product in order to save time, optimize the conditions for the PCR reaction and save the meager sample resources to be able to conduct amplification of gene fragments Two mastermixes finished-products were used in the study namely HotStarTaq Mastermix for samples with low DNA concentrations and DreamTaq Mastermix for amplification of samples with high DNA concentrations The total volume of each PCR reaction was 21 µl, consisting of 1-2 µl of DNA template depending on the quality of DNA in the final extraction solution, µl of each primer (10 µM/µl), µl water, 10 µl of Taq mastermix PCR conditions were: 95oC for 15min for Qiagen mastermix and 5’ for ThermoFisher mastermix; followed by 35 cycles, at 95oC in 30s, 48oC60oC in 45s, 72oC in 1min; final elongation step at 72oC in 6min Negative controls, each extraction and each PCR were carried out at the same time PCR products were visualized by electrophoresis through a 1% agarose gel, 2pg/ml ethidium-bromide, in 1X TBE buffer (Tris base, Boric acid, EDTA pH 8) at 90V for 30min After that, I was visualized by ultraviolet light on an Alphamager MINI (Protein Simple, USA) Successful PCR products were purified using the GeneJET PCR Purification kit (ThermoFisher Scientific, Lithuania) The procedure was performed according to the manufacturer's instructions including the following steps: attaching DNA to the membrane (using Binding Buffer and silica membrane filter column containing); clean DNA (using 350 µl Wash Buffer); Dissolve the DNA (using 30 µl of Elution Buffer) After purification, PCR products were stored at -4 oC and then sent for two-dimensional sequencing at First Base company (Malaysia) 2.3.6 Building phylogenetic tree The obtained sequences were aligned in Sequencher v5.4.6 software (Gene Codes Corp, Ann Arbor, MI, USA) The obtained sequences were checked using the Basic Local Alignment Search Tool (BLAST) available on the website of the National Center for Biotechnology Information The sequences were then solved together with those from the Gen Bank (Genbank) After the sequences were aligned using Clustal X v2 software (Thompson et al 1997), data were analyzed using maximum parsing (MP) as implemented in PAUP* 4.0b10 (Swofford 2001), maximum likelihood (ML) as implemented in IQ-TREE v1.6.7.1 (Nguyen et al 2015), and Bayesian Inference (BI) as implemented in MrBayes v3 2.7 (Ronquist et al 2012) The settings for MP and BI analyses followed Le et al (2006), except that the number of generations in Bayes analysis was increased to 1×107 Both BI and ML are run using a best-of-breed model, TVM+I+G, as chosen by jModelTest v2.1.4 (Posada 2008) The limition for memory function is 54 in Bayes analysis because the -lnL score reached a stable level after 54,000 generations in two times runs Nodal support was evaluated using 1,000 bootstrap replication (BP) as calculated in PAUP, 10,000 superfast bootstrap clones (UFB) in IQ-TREE v1.6.7.1 and posterior probabilities (PP) in IQ-TREE v1.6.7.1 MrBayes v3.2 BP ≥ 70 and PP, UFBP ≥ 95% were considered strong support for a clade (Hillis & Bull 1993; Ronquist et al 2012; Nguyen et al 2015) The uncorrected pairwise distance (p) were calculated in PAUP* 4.0b10 2.3.7 Statistical analysis The study used MS-Excel software in Microsoft Office 2010 to analyze data and compare the similarity in species composition between provinces with similar habitats in the North and Central regions based on data collected during the field survey and combined with the reference of published works In addition, in the study, the researcher also compared the correlation of species composition between regions, and used PAST Statistic software (Hammer et al 2001) for statistical analysis.The data was coded in symmetric form (1: Present; 0: Not present) The Sorensen-Dice index was calculated as follows: djk = 2M/(2M+N) M: the number of species recorded in two regions; N: the total number of species recorded in a region 2.3.8 Gecko distribution characters 1) Gecko species distribution along habitat types Based on the classification of vegetational cover types and the level of human impact on the vegetation according to the document “Guideline for biodiversity investigation and monitoring” (Pham Nhat et al 2003), and based on the current status of limestone forest in the studied area, the distribution of gecko species is divided into main habitat types including: - Limestone habitat in farming area (SC1): An area where people use land to grow agricultural and industrial crops or seasonal crops, including interlaced limestone ranges here (Fig 2.4) - Limestone habitat in secondary forest (SC2): A forest where it has been affected or not developed with few big trees, poor forest type, in which there is a distribution of limestone mountains (Fig 2.4) - Limestone habitat in primary forest (SC3): A forest that is low-impact rich with many big trees (Fig 2.4) 10 Chapter RESULTS AND DISCUSSION 3.1 Diversity of species composition of the Gecko family (Gekkonidae) Based on morphological and/or molecular analyses of 138 gecko specimens,28 gecko species of genera have been identified in the study areas consisting of species of the Bent-toed gecko (Cyrtodactylus); the Leaf-toed Gecko (Dixonius), House gecko (Hemidactylus), and Half leaf-fingered geckos (Hemiphyllodactylus) each of these has species; and Web-toed geckos (Gehyra) (Table 3.1) Table 3.1 List of Geckos (Gekkonidae) species recorded in study sites TT I II III IV V VI Name Scientific name Cyrtodactylus Cyrtodactylus houaphanensis*** Cyrtodactylus interdigitalis Cyrtodactylus muangfuangensis*** Cyrtodactylus ngoiensis*** Cyrtodactylus pageli Cyrtodactylus teyniei Cyrtodactylus wayakonei* Cyrtodactylus sp.1 Cyrtodactylus sp.2 Leaf-toed Gecko Dixonius Dixonius lao*** Dixonius siamensis Dixonius somchanhae*** Dixonius sp Gecko Gekko Gekko aaronbaueri Gekko kabkaebin Gekko khunkhamensis*** Gekko gecko Web-toed geckos Gehyra Gehyra mutilata Gehyra sp.1 Gehyra sp.2 House geckos Hemidactylus Hemidactylus bowringii Hemidactylus frenatus Hemidactylus ganotii Hemidactylus platyurus Half leaf-fingered Hemiphyllodactylus geckos Hemiphyllodactylus kiziriani* Hemiphyllodactylus fc.serpispecus Hemiphyllodactylus sp1 Hemiphyllodactylus sp2 VT Study sites LPB UDX XK HP KM Bent-toed Gecko + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Notes: (*): new species recorded for the province; (**): new species for science; (sp.): undetermined species; (cf.): closely related species Research location: VC= Vientiane, LPB= LuangPrabang, UDX=Udomxay, XK= Xiengkhoang, HP = Huaphane, KM = Khammuone Diversity of species: The genus Cyrtodactylus has the most diverse number of species with species, the genera as Leaf-toed Gecko (Dixonius), Gecko (Gekko), House geckos 11 (Hemidactylus), Half leaf-fingered geckos (Hemipyllodactylus) have species of each and Gehyra has species 3.1.1 New species discoveries - New species to science: Within the framework of this research, 06 new species have been described as following: 1) Cyrtodactylus muangfuangensis Sitthivong, Luu, Ha, Nguyen, Le & Ziegler, 2019 The phylogenetic tree also shows the new species as a sister species to C pageli, this species is also found in Vientiane province with detection sites about 50 km apart, morphologically, the new species may be clearly differentiated from C pageli in the number of ventral and anterior foraminal scales in both sexes In addition, the genetic difference between C muangfuangensis and C pageli was 18% based on a mitochondrial COI gene fragment (Fig 3.1) Figure 3.1 New species standard specimen Cyrtodactylus muangfuangensis (VNUF R.2018.32) male Photo: S Sitthivong 2) Cyrtodactylus houaphanensis Schneider, Luu, Sitthivong, Teynié, Le, Nguyen & Ziegler, 2020 Cyrtodactylus houaphanensis differs from all species in the genus, in the C wayakonei group having at least 3.3% genetic variation in the COI gene This new species is morphologically similar to C chauquangensis and is a sister taxon to C puhuensis by genetic analysis, and differs from the latter species by the absence of femoral fores (Fig 3.2) Figure 3.2 New species standard specimen Cyrtodactylus houaphanensis (IEBR A.2013.109) Male specimen Photo: A Teynie 3) Cyrtodactylus ngoiensis Schneider, Luu, Sitthivong, Teynié, Le, Nguyen & Ziegler, 2020 Cyrtodactylus ngoiensis differs from other closely related congeners by at least 11.6% genetic variation in COI genes The new species is believed to be a member of the C wayakonei species group, but is morphologically close to C dumnuii from Thailand (Fig 3.3) 12 Figure 3.3 New species Cyrtodactylus ngoiensis A: Male specimen (IEBR 4548); B: Female model (IEBR A.2013.110) Photos: A Teynié and T.Q Nguyen 4) Dixonius lao Nguyen, Sitthivong, Ngo, Luu, Nguyen, Le & Ziegler, 2020 In phylogenetic analyses, the new species was shown to be a sister taxon with two undescribed taxa from Thailand but differing by at least 8.6% in pairwise genetic distances with, the second is based on the complete sequence of the mitochondrial ND2 gene with partial or complete sequencing of six adjacent tRNAs (Fig 3.4) Figure 3.4 New species standard specimen Dixonius tuberculosis A: Male specimen (VNUF R.2016.2); B: Female model (IEBR A.2019.6) Photos: L Q Vinh 5) Dixonius somchanhae Nguyen, Luu, Sitthivong, Ngo, Nguyen, Le & Ziegler, 2021 Genetically, the new species and the closely related species D siamensis areapproximately 9.4% divergent from each other based on the complete mitochondrial ND2 gene with six partial or complete adjacent tRNAs It is the 12th known species of the genus Dixonius and the second Dixonius species to be described from Laos (Fig 3.5) Figure 3.5 New species Dixonius somchanhae A: Standard male specimen (VNUF R.2020.3); B: Male specimen (VNUF R.2020.2) Photos: L Q Vinh 6) Gekko khunkhamensis Sitthivong, Lo, Nguyen, Le, Ngo, Khotpathoom, Ziegler & Luu, 2021 This area is adjacent to Quang Binh province, Vietnam, where G scientiadventura was discovered New species differs from its congeners by at least 13% in pairwise distances based on a segment of the mitochondrial ND2 gene (Fig 3.6) Figure 3.6 New species Gekko khunkhamensis (A) Male specimen; (B) Female specimen Photos: S Sitthivong 3.1.2 New provincial records 1) Cyrtodactylus wayakonei Nguyen, Kingsada, Rosler, Auer & Ziegler, 2010 This species was originally described from Luang Nam Tha Province in Northern Laos in 2010, and in 2011 it was reported in Yunnan Province, China In this study, Cyrtodactylus wayakonei was encountered in Na Mo district, Udomxay Province, northern Laos The new sample has sequences close to the original description 13 Figure 3.7 (A) The holotype (IEBR A.2010.01); (B) New recorded specimen (VNUF R.2021.50) Photos: T.Q Nguyen and S Sitthivong 2) Hemiphyllodactylus kizirianii Nguyen, Botov, Le, Nophaseud, Zug, Bonkowski & Ziegler, 2014 Originally described in Luangprabang province in Northern Laos in 2014, in this study, the species Hemiphyllodactylus kiziriani was encountered in Namo district, Udomxay province, northern Laos The new sample has sequences close to the original description Figure 3.8 (A) The holotype (IEBR A.2014.3); (B) New recorded specimen (VNUF R.2021.52) Photos: T.Q Nguyen and S Sitthivong 3.1.3 Species morphological characteristics have not been identified In this study, 07 species have not been identified yet and are in the stage of molecular analysis, the species morphological characteristics are as followed: 1) Cyrtodactylus sp.1: Due to the lack of genetic comparison, the species has not been temporarily identified Figure 3.9 Unidentified specimen Cyrtodactylus sp.1 (A) Dorsolateral view of a female specimen; (B) A female specimen is molting Photos: S Sitthivong 2) Cyrtodactylus sp.2: Due to the lack of genetic comparison, the species has not been temporarily identified Figure 3.10 Unidentified specimen Cyrtodactylus sp.1 (A) Dorsolateral view of a male specimen; (B) Dorsal view of afemale specimen Photos: S Sitthivong 3) Gehyra sp.1: Because there has not been a genetic comparison, the species has not been accurately identified 14 Figure 3.11 Unidentified specimen Gehyra sp.1 (A) Dorsal view; (B) Ventral view Photos: S Sitthivong 4) Gehyra sp.2: Because there has not been a genetic comparison, the species has not been accurately identified Figure 3.12 Unidentified species Gehyra sp.2 (A) Dorsal side; (B) Ventral side Photo source: Saly Sitthivong 5) Dixonius sp Because there has not been a genetic comparison, the species has not been accurately identified Figure 3.13 Unidentified species Dixonius sp (A) Dorsal view; (B) Ventral view Photos: S Sitthivong 6) Hemiphyllodactylus sp.1: Due to the lack of genetic comparison, the species has not been temporarily identified Figure 3.14 Unidentified species Dixonius sp (A) Dorsal view; (B) Ventral view Photos: S Sitthivong 7) Hemiphyllodactylus sp.2: Because there has not been a genetic comparison, the species has not been accurately identified Figure 3.15 Unidentified species Hemiphyllodactylus sp (A) Dorsal view; (B) Ventral view Photos: S Sitthivong 15 3.1.4 Morphological characteristics of described gecko species in study sites 1) Cyrtodactylus interdigitalis Ulber, 1993 Head covered with small scales; prison muzzle; the eye is covered by the third eyelid; eardrum round, shallow, smaller than eye diameter; supralabials scales 9/9, infralabials scales 8/10; small, smooth dorsal scales; dorsal tubercle rows at midbody 16, row of ventral scales 34, precloacal pores absent in female 2) Cyrtodactylus pageli Schneider et al., 2011 Head covered with small scales; prison muzzle; the eye is covered by the third eyelid; eardrum round, shallow, smaller than eye diameter; supralabials scales 7-12, infralabials scales 7-9; dorsal tubercle rows at midbody 8-11, row of ventral scales 41-48, number precloacal pores 4-6 for (♂) and 4-6 for (♀), femoral foramen scale unknown; subdigital lamellae under whole fourth finger 19-23 and fourth toe 20-24 3) Cyrtodactylus teyniei David et al., 2011 Some morphological features: supralabials scales 9-13, infralabials scales 9-11; row of convex tubers on dorsal 17-22, row of scales surrounding body 112-138, row of ventral scales 34-42, number of scales precloacal pores: 8-14 for (♂) and 7-14 for (♀); subdigital lamellae under whole fourth finger 17-20 and fourth toe 18-22; dorsal and limbs brown 4) Dixonius siamensis Boulenger, 1899 Some morphological features: rows of ventral scales 22-26, supralabials scales 7-8, infralabials scales 6-9; row of dorsal convex tubercles 20-24, number precloacal pores scales: 7-8 for (♂) and absent in (♀); subdigital lamellae under whole fourth finger 10-12 Body small and slender, dark spots on body small and not round, rows of scales running along the body in alternating depressions 5) Gehyra mutilata Wiegmann, 1834 Some other common features: supralabials scales 610, infralabials scales 6-9; plates around chin plate 8-11, row of scales around body 101-134, row of ventral scales 36-43, precloacal pores scales: 36-42 for (♂) and absent in (♀); subdigital lamellae under whole fourth finger 6-8 and fourth toe 6-9, scale under caudal extended Specimen with identifiable characteristics consistent with Taylor's (1963) description: Scaled head; prison muzzle; the eye is covered by the third eyelid; round eardrum 6) Gekko aaronbaueri Ngo et al., 2015 Morphological characteristics: Head covered with small scales; prison muzzle; the eye is covered by the third eyelid; eardrum round, shallow, smaller than eye diameter; dorsal form with five to six dirty white vertebral spots interspersed with yellow, W-shaped spots between nape and spine and six to seven pairs of dirty white spots interspersed with dark spots with yellowish margins on the flanks between the extremities, the scales under the tail are extended 7) Gekko kabkaebin Grismer et al., 2019 Morphological features: Body has dark brown segments, separated by distinct wave-shaped black thread, light brown along the back The head is gray-brown, the nape has a W-shaped black streak The two tail edges are serrated, the eyes are black, the black line connects from the side of the neck running across the eye to the nose, the toes are connected by membranes 8) Gekko gecko Linnaeus, 1758 Pale greenish-gray back with yellow or bright red spots, with many nodules Males are more colorful than females The abdomen is white or gray with many small yellow dots The five-toed toe has claws and thin skin that forms a grip The eyes have vertical pupils, can expand in the dark, and have very good focus Living alone, only finding each other during the mating season 9) Hemidactylus bowringii Gray, 1845 On the dorsal and lateral surfaces, dark markings form thin longitudinal stripes with the lateral line most clearly defined; This latter stripe extends from the back of the eye to the shoulder, then is broken by circular cream dots, and a solid stripe at the base of the tail The dorsal stripe is always fragmented; begins on the front neck and extends to the base of the tail 16 10) Hemidactylus frenatus Duméril & Bibron, 1836 Usually gray or light brown to beige with green iridescence and white underside They also appear semi-transparent at times Their scales are usually uniform on the front, but increase in size along the back, and the large spiny scales are arranged in bands around the tail Has vertical pupils and is sensitive to darkness supralabials scales 9/9, infralabials scales 8/8, number of Subdigital lamellae under whole fourth finger 13, under fourth toe 15 11) Hemidactylus ganotii Duméril & Bibron, 1836 On the body is dark brown, tiny white dots on the body form a vertical line with the body The pupils of the eyes are brown and there is a black thread along with the eye, and the belly is yellow The flanks have alternating brown and white dotted stripes extending from the base of the hind thigh to the base of the front thigh, the scales under the tail are extended 12) Hemidactylus platyurus (Schneider, 1797) Morphological characteristics of the specimen are consistent with the previous description: Body length 50.1-59.4 mm, tail length 43.3-55.7 mm This gecko is easy to identify by the skin folds on the body from the armpit to the groin that is very clearly frilled, about 2mm wide at the widest place; the back of the thighs and shins also have narrow frills; body flattened, muzzle slightly pointed, head longer than wide, slightly differentiated from neck; round eye hole; triangular chin plate, with 10-11 supralabials scales and 8-9 infralabials scales 13) Hemiphyllodactylus cf serpispecus Eliades et al 2019 On the dark brown body, the black rings alternate in the middle, unevenly, the upper part of the middle of the tail base and the waist has brown marks The tail has brown patches interspersed with distinctly small black bands On the brown head, there are alternating black dots, with brown dotted stripes running from the neck to the eyes and over the eyelids 3.2 Genetic relationship of some genera in the Gecko family in Laos 3.2.1 Genetic relationship of the Bent-Toed Gecko (Cyrtodactylus) Most species of the genus Cyrtodactylus are distributed in limestone forests They are found on cliffs, rarely seen on the ground, there are a few species that are related to trees such as Cyrtodactylus interdigitalis and Cyrtodactylus buchardi Figure 3.30 Genetic tree of species in the genus Cyrtodactylus (Analysis (>50%), (-) unresolved, (*) represents a value of 100%) 17 The alignment contained no gaps MP analysis of the dataset recovered seven most parsimonious trees with 729 steps (CI = 0.5; RI = 0.78) In the ML analysis, the -Ln likelihood score of the best tree found was 3920.996 The topology derived from the BA was similar to those of Nguyen et al (2017) and Brennan et al (2017) Through the study, the genetic relationships of the genus Cyrtodactylus in Laos is divided into groups (Schneider et al 2020) such as: The first group is C wayakonei distributed in the North including species, the second group is C wayakonei distributed in the North, including species, the second group is C phongnhakebangensis distributed in the Central region includes 17 species and the last group is C irregularis distributed in the South including species 3.2.2 Genetic relationship of the genus Gekko Species of the genus Gekko are mainly distributed in limestone forests, often found on cliffs, only Tokay Gecko (Gekko gecko) species can live in many places including cliffs, on trees Farming areas are also available in people's houses from North to South Figure 3.32 Phylogenetic tree of species in the genus Gekko (-) unresolved node, (*) represents a value of 100% The alignment contained no gaps MP analysis of the dataset recovered seven most parsimonious trees with 1,296 steps (CI = 0.45; RI = 0.63) The topology derived from the BA was similar to those of Nguyen et al (2017) and Brennan et al (2017) (Fig 3.32) According to Wood et al (2021), the genus Gekko is divided into groups of Gecko (Gekko) in the world, including: Gekko (Gekko) gecko, Gekko (Japonigecko) japonicus, Gekko (Ptychozoom) homalocephala, Gekko (Phacogecko) rhacophorus, and Gekko (Sundagecko) vittatus, group Gekko (Pseudosundagecko) gulat and group Gekko (Archipelagecko) mindorensis In my research on the genus Gecko (Gekko) discovered species, new species for science, in groups including: group Gekko (Gekko) gecko with species Gekko gecko, group Gekko (Japonigecko) japonicus with species Gekko aaronbaueri and Gekko khunkhamensis, the group Gekko (Ptychozoom) homalocephala includes the species Gekko kabkaebin 18 3.2.3 Phylogenetic relationship of Leaf-toed Gecko (Dixonius) Species of the Dixonius are mainly distributed in secondary forests, cultivated areas, near populations with limestone mountains or large rocks on the ground, most species in this breed all live on the ground or cliffs close to the ground Figure 3.33 Phylogenetic tree of species in the genus Dixonius ((-) unresolved node, (*) represents a value of 100%) The alignment contained no gaps MP analysis of the dataset recovered seven most parsimonious trees with 1,433 steps (CI = 0.70; RI = 0.65) The topology derived from the BA was similar to those of Nguyen et al (2017) and Brennan et al (2017) Molecular data of D somchanhae from Laos, presented as sister taxon to D siamensis from Laos, Thailand and Vietnam with statistically significant values obtained from MP and BI analysis (Fig 3.33) 3.3 Evaluation of similarity in the family Gekkonidae composition in study sites 3.3.1 Among study sites Sorensen-Dice indexes among study sites are shown in Table 3.5 Table 3.5 Similarity index (Sorensen-Dice) composition among study sites Locations VC LPB UDX XK HP KM VC 0,454 LPB 0,300 UDX 0,666 0,600 XK 0,222 0,750 0,461 0,555 0,500 0,428 HP 0,444 0,421 0,352 0,521 KM 0,266 Note: VC= Vientiane Province, LPB= Luong Pha Bang Province, EK= Xieng Khuang Province, UDX= Udomxay province, HP= Huaphane Province and KM= Khammuone province Comparing the Sorensen-Dice index in Table 3.5, we see that Xiengkhoang province and Udomxay province have the highest similarity in gecko species composition with (djk = 0.750), followed by Luong Pha Bang and Udomxay province has a similar level of species composition of gecko species (djk = 0.666) because Udomxay province, Luangprabang province and Xieng Khuang province are all in the North with similar geographical borders, karst formations and ecosystems with limestone mountains and covered soil with many trees and forests 19 3.4 Distribution characteristics of Gecko species 3.4.1 Habitat distribution Based on the current status of karst forests in the study area, the distribution of gecko species in main habitat types has been assessed: (SC1) Limestone habitat in the cultivation area; (SC2) Limestone karst habitat in secondary forest; (SC3) Limestone mountain habitat in primeval forest The results are shown above (Figure 3.37) Hemiphyllodactylus Hemidactylus Gehyra Gekko Dixonius Cyrtodactylus 10 15 20 Limestone habitat in the cultivation area 25 Limestone karst habitat in secondary forest Limestone mountain habitat in primeval forest Figure 3.37 Gecko species diversity and varieties based on habitat in study sites 3.4.2 Altitude belt distribution Based on the collected, encountered and recorded specimens that have been synthesized in in figure 3.38 Species 6 4 4 4 33 3 2 2 11 1 1 1 1 0 Altitude (m) 1000 Hemiphyllodactylus Figure 3.38 Altitude belt distribution of Gecko species in stuy sites Research results showed that there are 11 species recorded at the altitude below 200 m, most of which are distributed here are common species in some genera such as Gekko and Hemidactylus, including species such as Dixonius siamensis; Gekko aaronbaueri; Gekko gecko; Gekko kabkaebin; Gehyra mutilata; Hemidactylus bowringii; Hemidactylus frenatus; Hemidactylus ganotii; Hemidactylus platyurus In which, two new species were discovered in this high altitude belt, such as Dixonius tubercle and Gekko khumkhamensis 20 3.4.3 Residence distribution The analysis results in 03 habitat types showed that the difference in the distribution of species is clear, especially in the habitat type per kilometer, there are few species, this can be explained because it is rare for species to live in trees It is not the main habitat of many species of geckos because only one species is recorded on the tree, the rest are common species and live in many forms (Fig 3.40) Species Individuals 83 21 34 26 ON THE CLIFF ON THE TREE ON THE GROUND Figure 3.40 Records on the habitat of gecko species in the study sites 3.5 Related problems of Gekko conservation in study sites 3.5.1 Rare and endemic species Precious and rare species in the study area account for a high proportion of up to 15 species (accounting for 30% of the total number of gecko species in Laos), up to 33 endemic species with a narrow distribution and are only distributed in Laos making up 66% of the total number of gecko species in Laos), there are 06 newly discovered species for science (occupying 12% of the total number of gecko species in Laos) Priority should be given to conserving their habitat forms and conducting more research sessions to search for more precious, rare and new species for science 3.5.2 Threat factors to Gecko species 1) Habitat loss and degradation Deforestation for swidden cultivation: Most of the forest area in the study area belongs to limestone mountains characterized by mountain peaks interspersed with valleys, so people often take advantage of arable land for reclamation swidden cultivation, especially in the Northern provinces because of the limestone mountains covered with soil and trees, people often take advantage of these areas Quarrying: Quarrying in the provinces of Laos is emerging a lot, especially in the provinces with many limestone mountains such as Khammuone province, Vientiane province, Huaphane province and Luangprabang province Roadworks: The road construction project in Huaphane province from Muong Hiem through Muong Xon to Pha Thi and Tu Muong Xon to the Laos-Vietnam border has destroyed a large area of natural forest at the multi-section road There are skewers in the Nam Et-Phu Loi National Park 21 Wildfires spread: In the dry season, from December to April next year, there are many areas of Laos where widespread forest fires occur during the survey period Logging and non-timber forest products: Talking about logging Lao PDR is very famous after the liberation of the country, in 1960 Laos covered about 71% of the country's area and by 2015 the coverage only about 40% remained, in the period 1980-2010 Laos' main income was from forest exploitation 2) Excessive hunting 3.5.3 Some proposed solutions for conservation work 1) Prioritized points for conservation The prioritied conservation area is determined based on the priority conservation point and the number of rare species present at that point Accordingly, the priority area for the conservation of gecko species has been identified, which is a high concentration of limestone mountains such as the Khammuone Province research area Cyrtodactylus jaegeri; Cyrtodactylus khammouanensis; Cyrtodactylus lomyenensis; Dixonius tuberculosis; Gekko aaronbaueri; Gekko boehmei; Gekko bonkowskii; Gekko khunkhamensis; Gekko sengchanthavongi; Gekko thakhekensis, Vientiane province includes: Cyrtodactylus muangfuangensis and Dixonius somchanha, Huaphane province: Cyrtodactylus houaphanensis, Luangprabang province has Cyrtodactylus ngoiensis and Udomxay Province has Cyrtodactylus wayakonei species (Fig 3.54) Figure 3.54 Prioritied conservation areas in the study sites 22 2) Prioritized species for conservation In the study area, there are a number of gecko species that are not on the list of rare and precious species, but are also being exploited strongly for export to Vietnam and China for food and medicinal wine, such as: Tokay Gekko Conservation activities also need attention to maintain and restore the population of this species 3) Priority conservation activities - Protect the habitat of geckos; - Propularize to raise awareness: - Develop to ecotourism: CONCLUSIONS - EXISTENCES - RECOMMENDATIONS Conclusions - The result has been recorded 28 species in genera of the Gecko family In which, the genus Cyrtodactylus has species; the genus Dixonius, Gekko, Hemidactylus, Hemiphyllodactylus have all species of each; and the genus Gehyra has species Specifically, 14 species belonging to genera have been recorded from Vientiane Province, species belonging to genera have been recorded from Luong Pha Bang Province, species belonging to genera have been recorded from Udomxay Province, species belonging to genera have been recorded from Xiengkhoang Province, 11 species belonging to genera have been recorded from Huaphane Province and 12 species of genera have been recorded from Khammuone Province Combined with previous studies bring, the total number of gecko species from Laosto 45 Notably, the thesis results have described to the new species of geckos to science from Laos, including: Cyrtodactylus houaphanensis, Cyrtodactylus muangfuangensis, Cyrtodactylus ngoiensis, Dixonius lao, Dixonius somchanhae and Gekko khunkhamensis Two species were recorded for the first time from Udomxay Province, including Cyrtodactylus wayakonei and Hemiphyllodactylus kiziriani There are potential species in the process of taxonomic analysis, including: Cyrtodactylus sp1, Cyrtodactylus sp2, Gehyra sp1, Gehyra sp2, Hemiphyllodactylus sp1, Hemiphyllodactylus sp2 Dixonius sp - The phylogenetic relationship of the genus Cyrtodactylus is divided into groups including C phongnhakebangensis, C wayakonei, and C irregularis; the genus Gekko has all belonging to the group Gekko japonicus; and the genus Dixonius has a member of the Dixonius siamensis group - The species composition of geckos between Xieng Khouang and Udomxay provinces has the highest degree of similarity (djk = 0.750), the lowest degree of similarity is from Vientiane to Xieng Khouang provinces (djk = 0.222), and between the Central and the North is (djk = 0.400) - Distribution characteristics: Distributed by habitat, habitat recorded the distribution of the most species of geckos with 22 species (accounting for 78.6%) Distributed by altitude belt, from 200 to less than 400 m altitude recorded the most species of geckos with 18 species (64.3% of total species), especially 06 new species for science were discovered in the high altitude belt Distribution by location, karst cliffs recorded the most with 26 species (92.9% of total species), species were recorded in two locations, no species was recorded in all locations 23 - Threats to gecko species and places that need to be prioritized for conservation: Deforestation for farming; quarry mining; forest fires; construction of transport infrastructure; harvesting timber and non-timber forest products; Hunted for food and for traditional medicine Regarding conservation status, 33 species are endemic to Laos, species are assessed as threatened in the Red List (IUCN, 2021) and 45 species are classified as Level II in Decree No 08/CP 2021 by the Government of Laos Places that need to be prioritized for conservation: The limestone mountains have a high concentration and distribution of rare and precious species such as the research sites in Vientiane, Luangprabang, Khammuone, Udomxay, Huaphane and Xiengkhoang provinces Existences - As for taxonomy, species have not been identified so far because DNA analysis is in progress and furthermorphological data is needed to support species identification - Since the study area has an average altitude of less than 800 m, most of the field studies are concentrated at an altitude of 150-650 m Moreover, the altitude above 800 m has dangerous terrain, difficult to access Therefore, there is little research data at altitudes above 800 m for comparison and evaluation - Regarding the conservation of gecko species, Decree No 08/CP/2021 of the Lao Government on the list of wild animals in Laos has criteria to evaluate and rank the priority of gecko species All species are listed in List II, this is due to the lack of a complete research database and the lack of experts on reptiles in general, geckos in particular Recommendations 1) The next research - Conduct additional research on species composition, especially unidentified species, conduct analysis and continue to investigate and collect more specimens - Conduct more research in the area with an altitude of over 800 m a.s.l., although the habitat is difficult, difficult to access, and the cool climate rarely encounters geckos but rare species 2) Proposals for conservation - Amending Decree No 08/CP/2021 of the Government on the list of Lao wild animals, the gecko species is based on legal and scientific basis to identify rare and precious species prioritized for protection, international documents including IUCN Red List and CITES Appendix - Focus on conserving locations with diverse species composition and habitats Implement conservation solutions for precious, rare and endemic species in priority conservation areas 24 LIST OF PUBLISHED ARTICLES Sitthivong, S., Luu, V.Q., Ha, N.V., Nguyen, T.Q., Le, M.D & Ziegler, T (2019) A new species of Cyrtodactylus (Squamata: Gekkonidae) from Vientiane Province, northern Laos Zootaxa, 4701 (3), 257-275 https://doi.org/10.11646/zootaxa.4701.3.3 Nguyen T.H., Sitthivong S., Ngo, H.T., Luu, V.Q., Nguyen, T.Q., Le, M.D & Ziegler, T (2020) A new species of Dixonius (Squamata: Gekkonidae) from the karst forest of Khammouane Province, central Laos Zootaxa, 4759 (4), 530-542 https://doi.org/10.11646/zootaxa.4759.4.4 Schneider, N., Luu, V.Q., Sitthivong, S., Teynié, A., Le, M.D., Nguyen, T.Q & Ziegler, T (2020) Two new species of Cyrtodactylus (Squamata: Gekkonidae) from northern Laos including new finding and expanded diagnosis of C bansocensis Zootaxa, 4822 (4), 503−530 https://doi.org/10.11646/zootaxa.4822.4.3 Sitthivong, S., Ha, V.N., Nguyen, H.T., Phimphasone, V., Nguyen, Q.T & Luu, V.Q (2020) New records of two gecko species (squamata: Gekkonidae) from Oudomxay province, Laos Journal of Forestry Science and Technology, 10 (2020), 96−104 Nguyen, T H., Luu, V Q., Sitthivong, S., Ngo, H T., Nguyen, T Q., Le, M D., & Ziegler, T (2021) A new species of Dixonius (Squamata: Gekkonidae) from Vientiane Capital, Laos Zootaxa, 4965 (2): 351-362 https://doi.org/10.11646/zootaxa.4759.4.4 Sitthivong S., Lo, O.V., Nguyen,T.Q., Ngo, H.T., Khotpathoom, T., Le, M.D., Ziegler, T & Luu, V.Q (2021) A new species of the Gekko japonicus group (Squamata: Gekkonidae) from Khammouane Province, central Laos Zootaxa, 5082 (6): 553-571 https://doi.org/10.11646/zootaxa.5082.6.3 ... (m) 23 0-6 84 23 2-6 75 62 0-8 27 64 2-7 82 34 8-7 02 29 8-4 39 1.08 0-1 .205 1.10 5-1 .397 16 8-5 24 17 0-2 14 68 7-1 .270 71 5-9 26 69 8-9 85 Time 25/ 7-9 /8/2018 23/1 0-6 /11/2018 21/ 4-2 8/4/2019 29/ 4-6 /5/2019 7/ 5-1 3/5/2019... 29/ 4-6 /5/2019 7/ 5-1 3/5/2019 14/ 5-2 0/5/2019 10/ 7-1 6/7/2019 17/ 7-2 3/7/2019 26/1 0-2 /11/2019 25/ 2-3 /3/2020 10/ 3-1 6/3/2020 17/ 3-2 3/3/2020 26/ 3-2 /4/2020 2.2 Research contents - Investigation of species... diameter; supralabials scales 7-1 2, infralabials scales 7-9 ; dorsal tubercle rows at midbody 8-1 1, row of ventral scales 4 1-4 8, number precloacal pores 4-6 for (♂) and 4-6 for (♀), femoral foramen