Phylogenetic analysis reveals cryptic species diversity within minute intestinal fluke,Stellantchasmus falcatus Onji and Nishio,1916(Trematoda,Heterophyidae)

Objective:To examine the taxonomic boundaries in the Stellantchasmus populations in different hosts and reveal the cryptic speciation of the Stellantchasmus in Thailand based on both morphological and molecular approaches.Methods:Stellantchasmus falcatus(S.falcatus) s.l.was collected from different hosts throughout Thailand.The worms were examined and measured.The nuclear ITS2 gene and the mitochondrial COI gene were used to investigate the S.falcatus s.l.from Dermogenus pusillus and Liza subviridis.Results:Stellantchasmus was one of food-borne trematode that widely distribute throughout Thailand.The comparison of S.falcatus s.l.from Dermogenus pusillus and Liza subviridis indicated a genetic divergence of nuD NA with 3.6% and mtD NA with 19.3%,respectively.Morphological characteristics were fairly different in the ratio of body size,length of prepharynx,and ratio of organ size.Conclusions:Our results provide initial evidence that S.falcatus s.l.from different hosts tend to be a different species based on both molecular and morphological characters.Cryptic species complexes are generally found among parasites that tend to have large populations and/or rapid evolution.The degree of genetic diversity existing would suggest the practice of targeted regimes to design and minimize these lections of anthelmintic resistance in the future.

The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci Complex in China

The sweetpotato whitefly Bemisia tabaci（Homoptera：Aleyrodidae）is a destructive pest of agriculture and horticulture worldwide.Recent phylogenetic analysis using mitochondrial cytochrome oxidase 1 sequences indicates that this whitefly is a species complex including at least 24 morphologically indistinguishable but genetically distinct cryptic species.In this study,the inter-species crosses of Middle East-Asia Minor 1（MEAM1）,Mediterranean（MED）and Asia II 7 cryptic species,which were referred to as B,Q and Cv biotypes before,were conducted in two different devices,leaf cages（7 cm3）and cylinder cages（280 cm3）,and at three temperatures of 22,30 and 38°C.Results indicated that no female progeny were produced in the reciprocal cross between MEAM1×Asia II 7,between MED×Asia II 7 cryptic species neither in leaf cage nor in cylinder cages,while 0.81 and 1.37% of females in the offspring were recorded in the reciprocal cross between MEAM1×MED in leaf cage experiments.Approximately 0.95-0.98% female progeny were recorded in the reciprocal cross between MEAM1×MED at 30°C,0.77% female progeny were recorded in the single cross direction between MEAM1 × MED at 22°C,and no female progeny were found in their reciprocal cross at 38°C in leaf cage.Our findings indicated that neither space dimension nor temperature have a significant effect on the hybridization of different B.tabaci cryptic species.

A paradox： rapid evolution rates of germline-limited sequences are associated with conserved patterns of rearrangements in cryptic species of Chilodonella uncinata(Protista, Ciliophora)

Some of the most extreme genome wide rearrangements are found in ciliates, which are unique in possessing both germline micronucleus and somatic macronucleus in every cell/organism. A series of DNA rearrangement events, including DNA elimination, chromosomal fragmentation, gene unscrambling and alternative processing, happen during macronuclear development. To assess the molecular evolution of macronuclear and germline-limited sequences in different cryptic species of Chilodonella uncinata, we characterized the actin, α-tubulin and β-tubulin genes in the micronucleus and macronucleus genomes of USA-SC2 strain and compared them with other strains(i.e. cryptic species). Three main results are:(i) rearrangement patterns between germline and soma are conserved for each gene among the cryptic species of C. uncinata;(ii) in contrast, the germlinelimited regions are highly divergent in sequence and length among the cryptic species;(iii) pointer shifting is frequent among the cryptic species. We speculate that pointer sequences may serve as the buffer between the conserved macronuclear destined sequences and rapidly-evolving internal eliminated sequences. The data combined with previous studies demonstrate the plasticity of gene rearrangement among different groups of ciliates and add to the growing data for the role of genome rearrangements in species differentiation.

Can incidental sighting data be used to elucidate habitat preferences and areas of suitable habitat for a cryptic species?

The involvement of communities in ecological studies has been shown to augment conservation efforts,especially for cryptic species.However,there is a lack of studies addressing the utility of incidental sighting records from community members in gaining knowledge on habitat preferences and distribution of suitable habitat for these organisms.This study compares preferences of the Lumholtz’s tree kangaroo(Dendrolagus lumholtzi;LTK),a cryptic rainforest folivore in northeastern Australia,for various habitat and climatic variables derived from data collected during scientific projects to those derived from incidental sighting records using ArcGIS and Maxent.Incidental sighting records suggest that the species uses a wider range of altitudes,annual rainfalls,annual mean temperatures and vegetation types than predicted by scientific studies.Incidental records also show that the species can persist in areas of lower rainfall during the wettest month and lower minimum temperature during the coldest month.Both data place the species within a comparable range of rainfalls during the driest month,maximum temperatures of the warmest month and soil types.When using identified preferences to assess the extent and distribution of suitable habitat,incidental records predicted more areas of suitable habitat than scientific records with an overlap of up to 91%between them.The present study proves that incidental sighting records can be a valuable part of the study of cryptic species and should be considered complementarily alongside scientific studies to obtain comprehensive ecological information of a species that can assist in its conservation.

Species Concepts as Applied to the Whitefly Bemisia tabaci Systematics:How Many Species Are There?

The worldwide distribution and extensive genetic diversity of the whitefly,Bemisia tabaci,has long been recognized.However,the levels of separation within B.tabaci and the nomenclature of the various genetic groups have been a subject of debate.Recent phylogenetic analyses indicate that B.tabaci is a complex composed of 28 morphologically indistinguishable species.In this article,we first review the debate and difficulties associated with B.tabaci＇s taxonomy and systematics,and argue for the need to apply the biological species concept in order to elucidate B.tabaci＇s systematics.We summarize the accumulated genetic and behavioural data on reproductive incompatibilities evident amongst phylogenetic mtCOI groups of B.tabaci.Crossing studies have been conducted with 14 of the 28 putative species covering 54 reciprocal inter-species pairs,and observations on mating behaviour have been conducted for seven species pairs.Data from both crossing trials and behavioural observations indicate a consistent pattern of reproductive isolation among the putative species.We then discuss the technical and conceptual complexities associated with crossing experiments and behavioural observations designed to reveal reproductive incompatibility.Finally,we elaborate on a strategy for further clarifying the pattern of reproductive isolation between B.tabaci groups and propose future research directions on the systematics of this complex.

Matrilineal History of the Rana longicrus Species Group(Rana,Ranidae,Anura) and the Description of a New Species from Hunan,southern China

Fieldwork in Jiemuxi National Nature Reserve, Hunan, China, discovered morphologically indis- tinguishable groups of brown frogs that bred at different, exclusive times of the year. A principal components analysis of morphometric data, molecular analyses, and the exclusive breeding season suggested the occurrence of two species. The population that breeds during the winter was found to be an undescribed species and was subsequently described as Rana jiemuxiensis sp. nov. The new cryptic species can be identified from its congeners at the same locality by having a different breeding season and a divergent DNA barcode, Six major lineages of brown frogs were resolved with high nodal support. Rana japonica, R. chaochiaoensis, R. omeimontis, and R. jiemuxiensis have independent maternal lineages. Rana hanlunica and R. maoershanensis have essentially identical maternal lineages and they appear to represent the same species. The validity of the species R. longicrus, R. zhenhaiensis, and R. culaiensis and their genealogical relationships are not resolved and deserve further study. The genealogy indicates that sympatric cryptic lineages probably widely exist in the R. longicrus group. This highlights the importance of future more fine-scaled samplings and the inclusion of nuclear genes.

Phylogenetic Relationships of the Genus Paramesotriton(Caudata: Salamandridae) with the Description of a New Species from Qixiling Nature Reserve, Jiangxi, Southeastern China and a Key to the species

The matrilineal genealogy of the genus Paramesotriton is hypothesized based on DNA sequences from mitochondrial NADH subunit two （ND2） and its flanking tRNAs （tRN^Arp and a partial tRNA^Ala）. The genealogy identifies a highly divergent, unnamed lineage from Qixiling Nature Reserve, Jiangxi, China and places it as the sister taxon of P. chinensis. The newly discovered population differs from other congeners by several features of external morphology including having large clusters of dark brown conical warts on the dorsum of the head, lateral surface of the body and dorsolateral ridges. Its intermittent dorsal vertebral ridge is the same color as other parts of the dorsum and tail narrows gradually from the base to the tip. Further, the new population differs from all congeners by an uncorrected P-distance of more than 9.38% in ND2. Consequently, we describe the new species of Asian warty newt （Salamandridae） as Paramesotriton qixilingensis sp. nov.

Integrative taxonomy of the Russet Bush Warbler Locustella mandelli complex reveals a new species from central China

Background: The Russet Bush Warbler Locustella(previously Bradypterus) mandelli complex occurs in mountains in the eastern Himalayas, southern China, Vietnam, the Philippines, and Indonesia. The taxonomy has been debated,with one(L. seebohmi) to four(L. seebohmi, L. mandelli, L. montis and L. timorensis) species having been recognised.Methods: We used an integrative approach, incorporating analyses of morphology, vocalizations and a molecular marker, to re-evaluate species limits in the L. mandelli complex.Results: We found that central Chinese L. mandelli differed from those from India through northern Southeast Asia to southeast China in plumage, morphometrics and song. All were easily classified by song, and(wing + culmen)/tail ratio overlapped only marginally. Both groups were reciprocally monophyletic in a mitochondrial cytochrome b(cytb) gene tree, with a mean divergence of 1.0 ± 0.2%. They were sympatric and mostly altitudinally segregated in the breeding season in southern Sichuan province. We found that the Mt Victoria(western Myanmar) population differed vocally from other L. mandelli, but no specimens are available. Taiwan Bush Warbler L. alishanensis was sister to the L. mandelli complex, with the most divergent song. Plumage, vocal and cytb evidence supported the distinctness of the south Vietnamese L. mandelli idonea. The Timor Bush Warbler L. timorensis, Javan Bush Warbler L.montis and Benguet Bush Warbler L. seebohmi differed distinctly in plumage, but among-population song variation in L. montis exceeded the differences between some populations of these taxa, and mean pairwise cytb divergences were only 0.5–0.9%. We also found that some L. montis populations differed morphologically.Conclusions: We conclude that the central Chinese population of Russet Bush Warbler represents a new species,which we describe herein, breeding at mid elevations in Sichuan, Shaanxi, Hubei, Hunan and Guizhou provinces.The taxonomic status of the other allopatric populations is less clear. However, as they differ to a degree comparable with that of the sympatric L. mandelli and the new species, we elevate L. idonea to species status, and retain L. seebohmi and L. montis as separate species, the latter with timorensis as a subspecies. Further research should focus on different populations of L. montis and the Mt Victoria population of L. mandelli.

Ceratopteris chunii and Ceratopteris chingii(Pteridaceae),two new diploid species from China,based on morphological,cytological,and molecular data

Understanding how natural hybridization and polyploidizations originate in plants requires identifying potential diploid ancestors.However,cryptic plant species are widespread,particularly in Ceratopteris(Pteridaceae).Identifying Ceratopteris cryptic species with different polyploidy levels is a challenge because Ceratopteris spp.exhibit high degrees of phenotypic plasticity.Here,two new cryptic species of Ceratopteris,Ceratopteris chunii and Ceratopteris chingii,are described and illustrated.Phylogenetic analyses reveal that each of the new species form a well-supported clade.C.chunii and C.chingii are similar to Ceratopteris gaudichaudii var.vulgaris and C.pteridoides,respectively,but distinct from their relatives in the stipe,basal pinna of the sterile leaf or subelliptic shape of the fertile leaf,as well as the spore surface.In addition,chromosome studies indicate that C.chunii and C.chingii are both diploid.These findings will help us further understand the origin of Ceratopteris polyploids in Asia.

A new species of genus Fejervarya (Anura:Dicroglossidae) from northern Thailand

We describe a new species of frog in the dicroglossid genus Fejervarya from Ban Monjong, Omkoi District, Chiang Mai Province, northern Thailand. Analysis of DNA sequence data from the mitochondrial gene 16S, advertisement calls, and morphological distinctiveness support recognition of the new species. Matrilineal genealogy suggests that the new population from Chiang Mai is a sister taxon to the South Asian clade that includes F. syhadrensis, F. granosa, and F. pierreL The new species, Fejervarya chiangmaiensis sp. nov., differs morphologically from its congeners by its relatively small body size and proportions and the presence of dorsal warts and dermal ridges. Discovery of this new species indicates that the biodiversity of amphibians in this region remains underestimated.

DNA barcoding of the family Sparidae along the coast of China and revelation of potential cryptic diversity in the IndoWest Pacific oceans based on COI and 16S rRNA genes

Sparids are of considerable economic importance in marine fishery and aquaculture in China, and the species diversity of this group is considered relatively high. However, the accurate species identification and delimitation of sparids in China remain unaddressed. In this study, we used mitochondrial cytochrome oxidase subunit ?(COI) and 16S ribosomal RNA(16S) genes to conduct DNA barcoding and species delimitation in eleven sparid species from the coastal waters of China. Based on Kimura-2 parameter genetic distances, the mean intraspecific/interspecific variation for COI and 16S were calculated as 0.004/0.152 and 0.002/0.072, respectively. All the conspecific individuals formed monophyletic clusters in neighbour-joining trees of both markers. An obvious barcoding gap was detected for each species, and a common genetic threshold of 1.3% sequence divergence was defined for species delimitation in both markers. Although the sequence variation of 16S was generally lower than that of COI, the results indicated that sparid species could be ef fectively and accurately identified and delimited by COI as well as 16S. Thus, we propose that the COI gene serve as the standard DNA barcode for sparids, and that the 16S gene could also be an ideal candidate barcode. Moreover, each of the six sparid species( Argyrops spinifer, Rhabdosargus sarba, Dentex hypselosomus, Acanthopagrus latus, Acanthopagrus australis and Acanthopagrus berda) showed high intraspecific divergence(>1.3% genetic threshold) with the remarkable geographic lineages in the Indo-West Pacific oceans, which supported that potential unrecognized cryptic species were in them. The potential cryptic diversity revealed here might be primarily attributed to the allopatric divergences caused by the long-term geographic isolation between the Indian and West Pacific oceans or between the opposite sides of the Indian Ocean. The results further suggest that a revision of taxonomic status of these species is required, followed by development of a biodiversity conservation strategy.

DNA barcoding and molecular phylogeny of Dumasia(Fabaceae:Phaseoleae)reveals a cryptic lineage

Dumasia taxonomy and classification have long been problematic.Species within this genus have few morphological differences and plants without flowers or fruits are difficult to accurately identify.In this study,we evaluated the ability of six DNA barcoding sequences,one nuclear(ITS)and five chloroplast regions(trnH-psbA,matK,rbcL,trnL-trnF,psbB-psbF),to efficiently identify Dumasia species.Most single markers or their combinations identify obvious barcoding gaps between intraspecific and interspecific genetic variation.Most combined analyses including ITS showed good species resolution and identification efficiency.We therefore suggest that ITS alone or a combination of ITS with any cpDNA marker are most suitable for DNA barcoding of Dumasia.The phylogenetic analyses clearly indicated that Dumasia yunnanensis is not monophyletic and is separated as two independent branches,which may result from cryptic differentiation.Our results demonstrate that molecular data can deepen the comprehension of taxonomy of Dumasia and provide an efficient approach for identification of the species.

Relationship of Old World Pseudoxenodon and New World Dipsadinae,with Comments on Underestimation of Species Diversity of Chinese Pseudoxenodon

Assessment of the relationship between Pseudoxenodon and Dipsadinae has been hampered by lack of adequate samples. In this paper, we conducted phylogenetic analyses using two mitochondrial genes （12S and 16S rRNA） and one nuclear gene （c-mos） from thirteen specimens representing two species of Pseudoxenodon, together with 84 sequences of caenophidians and an outgroup sequence of Boa constrictor. Our study suggests that the Southeast Asian genus, Pseudoxenodon forms a robust genetic subclade within South American xenodontines, indicating that at least one lineage within this genus entered or returned to the Old World （OW） from the New World （NW） across the Beringian Land Bridge during the early Tertiary and the warm mid-Miocene. It also reveals the high intraspecific genomic variation within the populations of Pseudoxenodon macrops, indicating that species diversity of Pseudoxenodon in China is likely underestimated.

DNA reveals long-distance partial migratory behavior in a cryptic owl lineage

Background: The Brown Hawk Owl complex is said to consist of three species,the Brown Boobook(Ninox scutulata),the Chocolate Boobook(Ninox randi) and the Northern Boobook(Ninox japonica),which includes the nominate migratory lineage Ninox japonica japonica and a mitochondrially distinct lineage of taxonomically uncertain status that has been recorded year-round at least in Taiwan.Overlap in ranges during migration and morphological similarity have led to difficulties in distinguishing the Brown Boobook from the Northern Boobook.Methods: PCR of cytochrome-b and Sanger sequencing of Ninox samples from Singapore and Brunei were used to determine sample identity.Results: Two out of four Singaporean samples and the Brunei sample were identified as Northern Boobooks.This is the first official record of this species in Singapore and represents a considerable range extension for the species.Further,the samples belong to the mitochondrially distinct lineage previously characterized in resident Taiwan Residents populations rather than to the well-known nominate migratory lineage.Conclusions: Our data show that the mitochondrial signature previously documented in resident Taiwan Residents populations of the Northern Boobook extends to migratory populations.This cryptic lineage may be more widespread in its breeding quarters,extending to the Chinese and Russian mainland,in which case the name florensis would apply to it.Further genetic and bioacoustic investigation is required to resolve the taxonomic status of this lineage.

Identifying Chinese species of Gammarus(Crustacea:Amphipoda) using DNA barcoding

Using a standard cytochrome c oxidase I sequence,DNA barcoding has been shown to be effective to distinguish known species and to discover cryptic species. Here we assessed the efficiency of DNA barcoding for the amphipod genus Gammarus from China. The maximum intraspecific divergence for widespread species,Gammarus lacustris,was 3.5%,and mean interspecific divergence reached 21.9%. We presented a conservative benchmark for determining provisional species using maximum intraspecific divergence of Gammarus lacustris. Thirty-one species possessed distinct barcode clusters. Two species were comprised of highly divergent clades with strong neighbor-joining bootstrap values,and likely indicated the presence of cryptic species. Although DNA barcoding is effective,future identification of species of Gammarus should incorporate DNA barcoding and morphological detection.

A New Species of Euphlyctis (Amphibia, Anura, Dicroglossidae) from the West Coastal Plains of India

The genus Euphlyctis is widely distributed across Southwestern Arabian Peninsula into parts of Southeast Asia. Five of the seven known Euphlyctis species are found within the Indian subcontinent. Here, we describe a new species, Euphlyctis karaavali sp. nov. from South-west coast of India, which was discovered during surveys engaging citizens. This species was identified to be distinct based on molecular and morphological evidence. We provide a detailed description of this species along with its call description and compare it with closest congeners. Previous studies in the region had identified this species as E. hexadactylus but suggested the possibility of it being cryptic. Genetically E. karaavali sp. nov. is distinct from E. hexadactylus with a genetic divergence of 9.2% （12S and 16S） and shows a high divergence with E. kalasgramensis and E. ehrenbergii （13.04% each）. Our findings are discussed in the context of cryptic species discovery, citizen engagement in scientific progress and conservation measures while suggesting future directions.

A Preliminary Phylogenetic Analysis of Luidia (Paxillosida: Luidiidae) from Chinese Waters with Cytochrome Oxidase Subunit I (COI) Sequences

Luidia Forbes （Paxillosida： Luidiidae） are common soft bottom sea stars with 49 described species. Because of substan- tial morphological diversity, the taxonomy of the genus is complex and hasn＇t been resolved definitely. In order to resolve general taxonomic issues, and determine species boundaries and phylogenetic relationships within the genus Luidia, the sequences of tyro- chrome oxidase subunit I （COI） gene from 24 specimens of Luidia, belonging to eight taxa in Chinese waters, were studied. Three sequences of two species in genus Luidia from GenBank were used to analyze the phylogenetie relationships. The molecular phy- logeny exhibited three main clades, each with strong bootstrap support： Clade A including Luidia quinaria from the Sea of Japan; Clade B including seven nominal species （L. quinaria von Martens, L. yesoensis Goto, L. ehangi Liu, Liao and Li, L. orientalis Fisher, L. avicularia Fisher, L. longispina Sladen and L. hardwicki Gray） from Chinese waters; and Clade C including L. maculata Miiller ＆ Troschel from Chinese waters. Our molecular phylogeny results support the morphological Quinaria-Group and Alter- nata-Group assigned by D6derlein. Seven nominal species we sampled do not exhibit genetic distances that are large enough to rec- ognize them as separate species. Cryptic species may exist in ＇Luidia quinaria＇ from the Yellow Sea and the Sea of Japan. Meaning- ful morphological characters need further investigation in Luidia.

Matrilineal Genealogy of Hynobius (Caudata:Hynobiidae) and a Temporal Perspective on Varying Levels of Diversity among Lineages of Salamanders on the Japanese Islands

Previous work found that different Japanese lineages of salamanders had quite different levels of species and genetic diversity. Lineages vary from having one to several species and the extent of genetic variation among lineages differs substantially. Most speciose,genus Hynobius contains 18 species and several potential cryptic species. We explore genetic diversity in this genus by combining comprehensive sampling and mitochondrial DNA sequences. Based on this and previous analyses of salamanders,relative times of divergence are employed to evaluate the relationship between age and diversity among the four major lineages whose distributions broadly overlap on the islands. For Hynobius,our analyses are congruent with the previously reported high level of cryptic diversity in morphology and allozymes,particularly in species composed of non-sister matrilines. Both species and genetic diversity correlate with the relative ages of the lineages. This correlation indicates that the variation in levels of diversity can be explained,to a considerable extent,by the hypothesis that older insular lineages have accumulated greater diversity. In addition to the Korean Peninsula,H. leechii might have survived in another Pleistocene glacial refugium north of the peninsula and this refugium provided a source of colonization after the last glacial maximum.

Impacts of climate warming on hybrid zone movement： Geographically diffuse and biologically porous ＂species borders＂

The ecology and evolutionary biology of insect-plant associations has real- ized extensive attention, especially during the past 60 years. The classifications （categorical designations） of continuous variation in biodiversity, ranging from global patterns （e.g., lat- itudinal gradients in species richness/diversity and degree of herbivore feeding specialization） to localized insect-plant associations that span the biospectrum from polyphenisms, polymorphisms, biotypes, demes, host races, to cryptic species, remain academically contentious. Semantic and biosystematic （taxonomical） disagreements sometimes detract from more important ecological and evolutionary processes that drive diversification, the dynamics of gene flow and local extinctions. This review addresses several aspects of insect specialization, host-associated divergence and ecological （including ＂hybrid＂） speciation, with special reference to the climate warming impacts on species borders of hybridiz- ing swallowtail butterflies （Papilionidae）. Interspecific hybrid introgression may result in collapse of multi-species communities or increase species numbers via homoploid hybrid speciation. We may see diverging, merging, or emerging genotypes across hybrid zones, all part of the ongoing processes of evolution. Molecular analyses of genetic mosaics and genomic dynamics with ＂divergence hitchhiking＂, combined with ecological, ethological and physiological studies of＂species porosity＂, have already begun to unveil some answers for some important ecological/evolutionary questions. （i） How rapidly can host-associated divergence lead to new species （and why doesn＇t it always do so, e.g., resulting in ＂incom- plete＂ speciation）？ （ii） How might ＂speciation genes＂ function, and how/where would we find them？ （iii） Can oscillations from specialists to generalists and back to specialists help explain global diversity in herbivorous insects？ （iv） How could recombinant interspecific hybridization lead to divergence and speciation？ From ancient phytochemically defined angiosperm affiliations to recent and very local geographical mosaics, the Papilionidae （swallowtail butterflies） have provided a model for enhanced understanding of ecological patterns and evolutionary processes, including host-associated genetic divergence, ge- nomic mosaics, genetic hitchhiking and sex-linked speciation genes. Apparent homoploid hybrid speciation in Papilio appears to have been catalyzed by climate warming-induced interspecific introgression of some, but not all, species diagnostic traits, reflecting strong divergent selection （discordant）, especially on the Z （= X） chromosome. Reproductive isolation of these novel recombinant hybrid genotypes appears to be accomplished via a delayed post-diapause emergence or temporal isolation, and is perhaps aided by the thermal landscape. Changing thermal landscapes appear to have created （and may destroy） novel recombinant hybrid genotypes and hybrid species.

Molecular phylogeographic analyses and species delimitations reveal that Leopoldamys edwardsi(Rodentia:Muridae)is a species complex

Leopoldamys edwardsi is a species with wide distribution ranges in southern China but is not discussed in stud­ies on geographic variation and species differentiation.We used 2 mitochondrial(Cytb,CO1)and 3 nuclear(GHR,IRBP and RAG1)genes to clarify species phylogeography and geographical differentiation.Maximum likelihood(ML)and Bayesian phylogenetic inference(BI)trees consistently indicated that L.edwardsi is a spe­cies complex containing 3 main lineages with high Kimura-2-parameter(K2P)divergences(i.e.lineages LN,LS and LHN)found in the northern and southern China and Hainan Island,respectively.The 3 species delimitation methods,automated barcoding gap discovery,Bayesian poisson tree process analysis and Bayesian phylogenet­ics and phylogeography,consistently supported the existence of cryptic species.Divergence times among the main lineages were inferred to be during the Pleistocene,with LHN/LS split at 1.33 Ma and LN/(LHN+LS)at 2.61 Ma;the diversifications of L.edwardsi complex might be caused by the rapid uplifts of Tibetan Plateau,paleo­climate change and complex topography.The divergence between LHN and LS was probably related to the sepa­ration of Hainan Island from the mainland via the formation of the Qiongzhou Strait.Lineages LN and(LS+LHN)likely diverged due to the Wuyi-Nanling mountain range forming a dispersal barrier.Our results suggested that L.edwardsi complex contains at least 3 distinct species:LHN represents L.hainanensis,endemic to Hainan Island and previously considered as a subspecies L.e.hainanensis;LS represents a cryptic species distributed through­out the southern Chinese continent;and LN represents the nominotypical species L.edwardsi.