Journal of Systematics and Evolution

Following the recent publication of global cicada phylogenetic trees by Marshall et al. (2018), ukasik et al. (2018), and Simon et al. (2019), we developed a new dated tree incorporating mostly endemic east Asian cicada data for totally 113 specimens, using the mostly advanced BEAST v1.X software applied the relaxed clock model. Fossil calibrations as old as Triassic were adopted after Moulds (2018), and a Quaternary geological event calibration was adopted following Osozawa et al. (2012), applying the calibration function of BEAST. Our timetree suggests that Tettigarctidae had cicada basal lineage as old as 200 Ma, and Derotettiginae was next as old as 100 Ma. Tibicininae was a sister of the resting Cicadidae, and Tettigomyiinae, Cicadettinae, and Cicadina started simultaneous branching and radiation around 40 Ma. We made a base substitution rate vs age diagram based on the timetree using the BEAST function, and it strongly suggested an exponential increase of base substitution rate approaching the present. The consequent increased cicada biodiversity including generation of cryptic species might have been driven by the generation and spreading of C4 grasses and the following Quaternary glaciations and severe environmental change.

The enigmatic monotypic genus Paroxygraphis (Ranunculaceae, trib. Ranunculeae), represented solely by P. sikkimensis, has long posed taxonomic uncertainties due to its rarity. Through field rediscovery in southern Xizang province, China, and comprehensive molecular phylogenetic analyses combining nrITS and four plastid regions, we resolve its phylogenetic position within the Oxygraphis clade, with an estimated divergence time of about 3.73 Ma (95% HPD: 1.98-5.77 Ma) from its sister clade. Morphological and ecological characteristics, including small herb with a rosette of basal simple leaves, longitudinally ribbed achenes, and its alpine adaptations, further support this relationship. Furthermore, the results showed that persistent sepals and unisexual flowers in trib. Ranunculeae reflects multiple independent evolutionary origins. We propose the new combination Oxygraphis sikkimensis and highlight its critically endangered status due to habitat vulnerability. Lectotypification of P. sikkimensis is also made. This study provides new data for the floristic research of China and resolves the long-standing taxonomic controversy of this problematic genus within trib. Ranunculeae.

Target enrichment and RAD-seq are well-established high throughput sequencing technologies that have been increasingly used for phylogenomic studies, and the choice between methods is a practical issue for plant systematists studying the evolutionary histories of biodiversity of relatively recent origins. However, few studies have compared the congruence and conflict between results from the two methods within the same group of organisms, especially in plants, where extensive genome duplication events may complicate phylogenomic analyses. Unfortunately, currently widely used pipelines for target enrichment data analysis do not have a vigorous procedure for remove paralogs in Hyb-Seq data. In this study, we employed RAD-seq and Hyb-Seq of Angiosperm 353 genes in phylogenomic and biogeographic studies of Hamamelis (the witch-hazels) and Castanea (chestnuts), two classic examples exhibiting the well-known eastern Asian-eastern North American disjunct distribution. We compared these two methods side by side and developed a new pipeline (PPD) with a more vigorous removal of putative paralogs from Hyb-Seq data. The new pipeline considers both sequence similarity and heterozygous sites at each locus in identification of paralogous. We used our pipeline to construct robust datasets for comparison between methods and downstream analyses on the two genera. Our results demonstrated that the PPD identified many more putative paralogs than the popular method HybPiper. Comparisons of tree topologies and divergence times showed significant differences between data from HybPiper and data from our new PPD pipeline, likely due to the error signals from the paralogous genes undetected by HybPiper, but trimmed by PPD. We found that phylogenies and divergence times estimated from our RAD-seq and Hyb-Seq-PPD were largely congruent. We highlight the importance of removal paralogs in enrichment data, and discuss the merits of RAD-seq and Hyb-Seq. Finally, phylogenetic analyses of RAD-seq and Hyb-Seq resulted in well-resolved species relationships, and revealed ancient introgression in both genera. Biogeographic analyses including fossil data revealed a complicated history of each genus involving multiple intercontinental dispersals and local extinctions in areas outside of the taxas modern ranges in both the Paleogene and Neogene. Our study demonstrates the value of additional steps for filtering paralogous gene content from Angiosperm 353 data, such as our new PPD pipeline described in this study. [RAD-seq, Hyb-Seq, paralogs, Castanea, Hamamelis, eastern Asia-eastern North America disjunction, biogeography, ancient introgression]

Background--Major northern temperate tree genera emerged in the Eocene and now have vast ranges across the Northern Hemisphere. Here we undertake a multi-discipline study to provide novel insights into the formation and biogeographic history of a dominant, yet understudied temperate tree genus, Fagus L. Data and methodology--A whole chloroplast genome phylogeny (eighty-two chloroplast genomes) with multi-accessions of most species was determined and contrasted with the timing of lineage diversification inferred from three different nuclear and chloroplast gene time-calibrated phylogenies using 101 fossils as age priors. Main results--Five deeply diverged chloroplast lineages were revealed that, with the exception of a Eurasian lineage, underwent divergence decoupled from speciation predating modern species lineages by up to 28 million years (Ma). The geographic distribution of chloroplast lineages reflects a complex history of genetic admixture during past contact between extinct and modern species including between different subgenera in East Asia and long-term persistence particularly in Japan which is the hotspot of plastome diversity. Conclusion--Modern Fagus forests are the consequence of 62 million years of evolution, migration, genetic exchange and species extinction with most modern species harbouring a mosaic of genetic material, a legacy of multiple admixture events in deep time.

Germplasm resource is the source of herbal medicine production. Cultivation of superior germplasm resources helps to resolve the serious conflict between long-term population persistence and growing market demand by producing materials with high quality consistently. Cibotium barometz is the original plant of cibotii rhizoma ("Gouji"), a traditional Chinese medicine used in the therapy of pain, weakness, and numbness of lower extremity. Long-history use of Cibotium has rendered wild populations of this species declined seriously in China. Without sufficient understanding of species and lineage diversity of Cibotium, it is difficult to propose a targeted conservation scheme at present, let alone selecting high-quality germplasm resources. In order to fill such a knowledge gap, this study sampled C. barometz and relative species throughout their distribution in China, performed genome skimming to obtain plastome data, and conducted phylogenomic analyses. We constructed a well-supported plastome phylogeny of Chinese Cibotium, which showed that three species with significant genetic difference distributed in China, namely C. barometz, C. cumingii, and C. sino-burmaense, a cryptic species endemic to NW Yunnan and adjacent region of NE Myanmar. Moreover, our results revealed two differentiated lineages of C. barometz distributed in the east and west side of a classic phylogeographic boundary that probably shaped by monsoons and landforms in China. We also evaluated the resolution of nine traditional barcode loci, and designed five new DNA barcodes based on the plastome data which can discriminate all these species and lineages of Chinese Cibotium accurately. These novel findings integrated genetic basis will guide conservation planners and medicinal plant breeders to build systematic conservation plans and exploit germplasm resources of Cibotium in China.

O_LIThe expansive Himalayan-Hengduan Mountains (HM) and Qinghai-Tibet Plateau (QTP) form a biodiversity hotspot imperiled by global change. The species diversity, resulting from myriad factors led to plant diversification from 10 million years ago till now. However, despite understanding speciation timing better, a lack of in-depth population-level studies and a dearth of organismal sampling among closely related species and populations leaves an incomplete view of diversification trends and biotic influences. C_LIO_LIWe delve into the complex factors influencing diversification through genomic and eco-morphological analysis within the Stellera chamaejasme L. complex, known for its significant floral diversity. C_LIO_LIOur results uncover four cryptic species, indicating a more recent and younger diversification period ([~]2.67-0.9 Mya), driven by Pleistocene glaciation and a complex set of interacting biotic factors. These factors prompted allopatric speciation and advocated cyclical warming-cooling episodes along latitudinal and altitudinal gradients throughout the Pleistocene. C_LIO_LIThe study emphasizes the existence of cryptic species within these mountains, broadening our understanding of species diversification to more recent than previously recognized. This novel perspective may reshape evolutionary paradigms in plant science. Additionally, it also brings to light concerns over future warmings possible impacts on alpine species distribution and speciation, underlining the urgency for increased conservation efforts. C_LI

Recent genomic studies have suggested that hybridization may play a significant role in adaptive radiation, rapid speciation, and convergent evolution. The genus Oxera, a plant taxon thought to have diversified at its beginning through adaptive radiation in New Caledonia, provided an opportunity to investigate these processes. Within the robusta subclade of Oxera, characterized by bird-pollinated yellow-orange flowers, convergent evolution of flower shape is likely to have occurred. We aimed to elucidate the hybridization history of the robusta subclade by whole genome sequencing and MIG-seq data. Our analyses revealed an ancestral introgression from O. coriacea to O. sympatrica, whose flowers are remarkably similar to each other. Among the introgressed genomic regions, we identified several genes potentially involved in flower shape development. O. sympatrica and its sympatric sister species exhibit distinct flower shapes, and pollinator-mediated reproductive isolation presumed to be a major barrier between them. The ancestral introgression uncovered in this study may have driven the convergent evolution of flower shape in the robusta subclade and played a crucial role in the speciation process of O. sympatrica. These finding contribute to our understanding of the interplay between hybridization, adaptive radiation, and speciation process.

Theaceae, with three tribes and nine genera, is a family of great economic and ecological importance. Recent phylogenetic analyses based on plastid genome resolved the relationship among three tribes and the intergeneric relationships within Gordonieae and Stewartieae. However, generic level relationships within the largest tribe Theeae were not fully resolved and potential hybridization among genera within Theeae revealed previously also remains to be tested further. Here we conducted a comprehensive phylogenomic study of Theaceae based on transcriptomes and low-depth whole-genome sequencing of 57 species as well as additional plastome sequence data from previous work. Phylogenetic analyses suggested that Stewartieae was the first-diverging clade in Theaceae, consistent with previous study using plastomic data. Within Theeae, the highly supported Apterosperma-Laplacea clade grouped with Pyrenaria with maximum support based on the partitioned and unpartitioned concatenation analyses using the 610 low-copy nuclear genes, leaving Camellia and Polyspora as another sister genera in the tribe. PhyloNet analyses suggested one reticulation event within Camellia and Pyrenaria respectively, but no intergeneric reticulations were detected in Theeae. Another introgression was found between Gordonia lasianthus and the common ancestor of Gordonieae during the Late Oligocene. The existing land bridges (e.g. Bering land bridge) might have facilitated this ancient introgression. Further researches need to be conducted to uncover the interspecific introgression pattern within Camellia. Ks distribution analyses supported the tea family shared one whole-genome duplication (WGD) event Ad-{beta}, which was recently mapped to the clade containing core Ericales, Primuloids, Polemonioids and Lecythidaceae.

Although the possibility of introgression from ghost lineages (all unsampled extant and extinct taxa) is now widely recognized, detecting and characterizing ghost introgression remains a challenge. Here, we propose a combined use of the popular D-statistic method, which tests for the presence of introgression, and the full-likelihood method BPP, which determines which of the possible gene-flow scenarios, including ghost introgression, is truly responsible. We illustrate the utility of this approach by investigating the reticulation and bifurcation history of the genus Carya (Juglandaceae), including the beaked hickory Carya sinensis. To achieve this goal, we generated two chromosome-level reference genomes respectively for C. sinensis and C. cathayensis. Furthermore, we re-sequenced the whole genomes of 43 individuals from C. sinensis and one individual from each of the 11 diploid species of Carya. The latter dataset with one individual per species is used to reconstruct the phylogenetic networks and estimate the divergence time of Carya. Our results unambiguously demonstrate the presence of ghost introgression from an extinct lineage into the beaked hickory, dispelling certain misconceptions about the phylogenetic history of C. sinensis. We also discuss the profound implications of ghost introgression into C. sinensis for the historical biogeography of hickory species. [BPP; Carya; D-statistic; gene flow; ghost introgression]

O_LITraditional classification of speciation modes has focused on physical barriers to gene flow. While allopatry has been viewed as the most common mechanism of speciation, parapatry and sympatry, both entail speciation in the face of ongoing gene flow and thus both are far more difficult to detect and demonstrate. Iberodes (Boraginaceae, NW Europe) with a small number of recently derived species (five) and contrasting morphological traits, habitats and distribution patterns constitutes an ideal system in which to study drivers of lineage divergence and differentiation. C_LIO_LITo reconstruct the evolutionary history of the genus, we undertook an integrative study entailing: (i) phylogenomics based on restriction-site associated DNA sequencing (RAD-seq), (ii) morphometrics, and (iii) climatic niche modelling. C_LIO_LIKey results revealed a history of repeated progenitor-derivative speciation, manifesting in paraphyletic pattern within Iberodes. Climatic niche analyses, together with the morphometric data and species distributions, suggest that ecological and geographical differentiation have interacted to shape the diversity of allopatric and parapatric distributions observed in Iberodes. C_LIO_LIOur integrative study has enabled to overcome previous barriers to understanding parapatric speciation by demonstrating the recurrence of progenitor-derivative speciation in plants with gene flow and ecological differentiation, explaining observed parapatry and paraphyly. C_LI

A total of 212 melon accessions with diverse geographical origins were classified into large and small seed-types by length of seed at the boundary of 9 mm, and into five populations based on polymorphisms in the nuclear genome. They were further divided into three maternal lineages, Ia, Ib, and Ic, by polymorphisms in the chloroplast genome. By combining these three classifications, the Europe/US subsp. melo and the East Asian subsp. agrestis were characterized as [large seed, Ib, PopA1 or A2] and [small seed, Ia, PopB1 or B2], respectively, indicating nearly perfect divergence in both nuclear and cytoplasm genomes. In contrast, in South and Southeast Asia, in addition to the Europe/US and East Asian types, recombinant types were also frequently found, indicating unclear genetic differentiation in South and Southeast Asia. Such an intermixed structure of genetic variation supported the Indian origin of Ia and Ib types of melon. Seed length was intermediate, between the large and small seed-types, and chloroplast type was a mixture of Ia and Ib in Momordica, suggesting its origin from the recombinant type. In Africa, three lineages of melon were distributed allopatrically and showed distinct divergence. Subsp. agrestis of the Ic type proved to be endemic to Africa, indicating its African origin.

Due to global climate cooling and aridification since the Paleogene, the members of the Neogene flora were extirpated from the Northern Hemisphere or were confined to a few refugial areas. For some species, the final reduction/extinction came in the Pleistocene, but some others have survived climatic transformations up to the present. This has occurred in Castanea sativa, a species of high commercial value in Europe and a significant component of the Caucasian forests biodiversity. In contrast to the European range, neither the historical biogeography nor the population genetic structure of the species in the isolated Caucasian range has been clarified. Here, based on a survey of 21 natural populations from the Caucasus and a single one from Europe, we provide likely biogeographic reconstruction and genetic diversity details. By applying Bayesian inference, species distribution modelling, and fossil pollen data, we estimated (1) the time of the Caucasian - European divergence during the Middle Pleistocene (436.5 ka), (2) the time of divergence among Caucasian lineages, and (3) outlined the glacial refugia for species. The climate changes related to the Early Middle Pleistocene Transition and the alpine orogenic uplift in the region are proposed as the major drivers of the intraspecific divergence and European-Caucasian disjunction, while the impact of the last glacial cycle was of marginal importance.

Premise of the studyWith over 3100 species, Astragalus (Fabaceae) has long fascinated botanists as the largest genus of flowering plants. With an origin in the Middle Miocene, Astragalus has one of the highest diversification rates known in flowering plants. Comprehensive taxonomic treatments exist, and the genus is currently subdivided into 136 sections in the Old World and 93 sections in the New World based on morphological characters. Despite considerable efforts, a comprehensive and well-resolved phylogeny of the genus is still lacking. MethodsHere, we reconstruct the backbone phylogeny of Astragalus using a custom bait set capturing 819 loci specifically designed for a target enrichment approach in the Astragalean clade. We carefully selected a set of 107 taxa representing all major clades currently recognized in Astragalus. Of those, 80 newly sequenced taxa were obtained from herbarium specimens as old as 110 years. Key resultsWe retrieved all the targeted loci and additional off-target plastome sequences for all samples, including the 80 herbarium specimens. Our phylogenetic analysis reinforced the currently accepted backbone phylogeny of Astragalus with high support and unprecedented details, additionally providing insights into cytonuclear phylogenetic conflicts in the genus. Evidence for potential reticulate evolution was found, providing a possible explanation for the conflicts observed. ConclusionsThis work represents an important milestone in obtaining a comprehensive, herbarium-based phylogeny of Astragalus, which will constitute the base to study a wealth of relevant biological questions, for example, the still unanswered question of what drove the rapid diversification of Astragalus, with important repercussions on our understanding of diversification in natural contexts.

Mating systems play a crucial role in plant speciation. In particular, hybrid seed inviability is prevalent among species with different mating systems due to parental conflict for resource allocation to offspring. However, whether such a postzygotic barrier can be rapidly established in recently diverged species remains poorly understood. In this study, we integrate population genomic and ecological approaches to address this question using recently diverged species pairs Aquilegia kansuensis and A. ecalcarata, which diverged approximately 0.256 Ma, in a sympatric population from Qinghai, China. Population structure and demographic history results reveal clear genetic differentiation between the two species, corroborating their recent divergence. The results of outcrossing rate estimation based on microsatellite markers indicated that A. kansuensis and A. ecalcarata exhibit high selfing and mixed mating systems, respectively. We performed reciprocal crosses between A. kansuensis and A. ecalcarata and found that A. ecalcarata yielded a very small number of small-sized seeds when selfed A. kansuensis was used as pollen donors, resulting in strong asymmetric reproductive isolation. An approximate Bayesian computation framework identified that approximately 4.6% of genomic loci are associated with reproductive isolation, and gene ontology analyses of these loci highlighted key pathways involved in seed maturation and post-embryonic development. Therefore, our findings provide evidence that A. ecalcarata and A. kansuensis represent a novel case of parental conflict and postzygotic isolation driven by divergent mating systems, suggesting mating systems can play a critical role in rapid plant speciation.

Phylogenetic relationships for the genus Calliandra section Androcallis (Fabaceae) were reconstructed, including previously sequenced species from Central and South America and unexamined species from Colombia, one of Calliandra main diversity centers. Here, we generated novel DNA sequences of Calliandra species from Colombia for the nuclear Internal Transcribed Spacer (ITS) and the chloroplast trnL and trnL-F intergenic spacer. By incorporating a broader taxonomic sampling, the relationships among main clades in Androcallis were clarified, providing a systematics framework in which to test evolutionary hypotheses. Phylogenetic analysis recovered five well-supported clades within Androcallis. Most species within each clade had similar geographical distributions and relationships between the five major clades are strongly supported for the first time. However, core Androcallis relationships, including most species from Colombia sequenced here, remain unclear. A second goal of this study was to determine the taxonomic status of Calliandra medellinensis. This enigmatic taxon emblematic of Medellin, Colombia, is found in limited numbers within the Aburra Valley and has been proposed to be a hybrid taxon. Here, C. medellinensis, C. magdalenae and C. haematocephala were not monophyletic within the core Androcallis clade. This suggests that C. medellinensis could potentially be an interspecific hybrid between C. magdalenae and C. haematocephala, thus challenging the taxonomic status of this species; however, more informative molecular markers should be used in future studies. Specifically, genomic studies should assess interspecific hybridization demographic models. Such insights can illuminate the C. medellinensis origin, guiding conservation strategies and providing valuable evolutionary overviews.

The central-marginal hypothesis (CMH) predicts high genetic diversity at the species geographic centre and low genetic diversity at the species geographic margins. However, most studies examining the CMH have neglected potential effect of past climate. Here, we test six hypotheses, representing effects of past climate and contemporary range position, for their ability to explain the spatial patterns of genetic diversity in 37 populations of Betula albosinensis. Ecological niche modelling (ENM) revealed large and continuous suitable habitats in north, southwest and southeast China during the last glacial maximum (LGM) but a contraction of suitable habitats since the LGM. Pollen records further confirmed the existence of multiple refugia in north and south China. The spatial pattern of genetic diversity (i.e., expected heterozygosity, gene diversity and allele richness) were best explained by distance to the southern edge and distance to the range edge but also showed longitudinal and latitudinal gradients. Hypotheses accounting for the effects of climate (climatic suitability, climatic stability and climatic variability) had comparatively little support. Our findings show partial support for the CMH and illustrates that the existence of multiple LGM refugia, and suggests species abundance and past species movement play a role in shaping genetic diversity across species ranges.

Elucidating the origin of carpel has been a challenge in botany for a long time. The Unifying Theory suggested that the carpel originate from a composite organ comprising an ovule-bearing shoot and a foliar part enclosing the shoot. A logical inference from this theory is that placenta in angiosperms should have radiosymmetrical vasculature, just like that in a young branch. Anaxagorea is the most basal genus of the primitive angiosperm family, Annonaceae. The conspicuous carpel stipe makes it an ideal material for exploring the carpel vasculature. In this study, serial sections of flower and carpel were delineated in Anaxagorea luzonensis and A. javanica, and a three-dimensional model of the carpel vasculature was reconstructed. The results show that (1) vascular bundles at both the carpel stipe and the ovule/placenta are in a radiosymmetrical pattern, (2) the amphicribral bundles would develop into ring-arranged bundle complex with the carpel maturation, (3) the ovule/placenta bundles were separated from the bundles of the carpel wall, and, (4) all the radiosymmetrical vasculature (including amphicribral bundles and ring-arranged bundle complexes) in the carpel were fed by a larger radiosymmetrical bundle system. These results suggest that the radiosymmetrical pattern of carpel vasculature are in line with the Unifying Theory.

Meiogyne is a genus of trees and treelets occurring in Indomalaya and Australasia-Pacific, an unusually wide distribution across Australasia and Western Pacific compared to the rest of the family Annonaceae. Previous chloroplast phylogenies of the genus offered poor resolution and support for many internal nodes. Here, a molecular phylogeny was reconstructed based on seven chloroplast and 11 nuclear markers of 33 Meiogyne taxa (ca. 70% sampling). The combined dataset generated a well resolved and supported phylogeny. Estimation of divergence time was calibrated with two fossils using uncorrelated lognormal relaxed clock model. Trait-dependent and trait-independent biogeographical models in BioGeoBEARS were compared using AICc weight and likelihood ratio test. The results suggest that narrow monocarp width and fruit colour associated with bird dispersal are correlated with increased macroevolutionary dispersal. Under the best-fitting monocarp width-dependent DEC model, a single colonisation event from Sunda to Sahul during the middle to late Miocene and two dispersal events from New Guinea and Australia into the Pacific during the late Miocene to early Pliocene were detected. BayesTraits analysis strongly supports a correlation between narrow fruits and avian fruit colours. This study reveals that Meiogyne lineages with narrow fruitlets and fruit colour associated to bird dispersal (black, red & orange) are associated with increased macroevolutionary dispersal. Bird dispersal and the associated traits may be important drivers for macroevolutionary dispersal for plants with fleshy fruits in Australasia-Pacific.

In northeast Asia, substantial portion of the floras, including endangered species, are shared among its component countries in the continental, peninsula, and island parts largely through Quaternary migration. To effectively conserve nationally endangered plants in Northeast Asia, transnational conservation studies are vitally needed. Lychnis wilfordii (Caryophyllaceae) has disjunct distribution in Russian Far East (Primorsky Krai), northeast China (Jilin), Korea (Gangwon-do) and Japan (Hokkaido, Aomori, Nagano), surrounding the sea, and this is designated as an endangered species in Japan and Korea. Population genetic and molecular dating analyses were conducted 1) to elucidate geographic genetic structure covering the species range, 2) to test possible scenarios of migration, and 3) to develop logical plans for effective conservation. Population genetic analyses indicated the continent and peninsula parts (north and south Primorsky Krai, Jilin, and Gangwon-do) had higher genetic diversity compared to those in the Japanese Archipelago (Hokkaido and Nagano). Five genetically distinct groups were recognized, namely, Nagano, Gangwon-do, Jilin, north and south Primorsky Krai plus Aomori, and Hokkaido. Genetic distance between Hokkaido and Nagano was larger than between Hokkaido and north Primorsky Krai, and between Nagano and Gangwon-do, crossing national borders and the natural barrier of the sea. Considering these results, L. wilfordii likely migrated from the Asian continent to the Japanese Archipelago using two routes: north route from Russian Far East to Hokkaido and Aomori, and south route from the Korean Peninsula to Nagano. Based on molecular dating, migration from the continent to the islands likely occurred from the middle Pleistocene to the Holocene. For effective conservation of L. wilfordii, Hokkaido and Nagano populations should be distinguished as different evolutionary significant units, although these two regions belong to the same country, because Hokkaido and Nagano populations are at the different ends of the two migratory routes based on the migration scenario.

The sky islands of Southwest China, characterized by dramatic topographical and climatic variations, are prominent hotspots of biodiversity and endemism. Organisms inhabiting middle-to-high elevation habitats in this region are geographically isolated within distinct mountain chains, which over geological time have been subjected to isolation-by-distance and isolation-by-environment. These processes have led to profound allopatric diversification and strong phylogeographic structuring, resulting in a plethora of genetically distinct cryptic species, as is becoming increasingly evident for many small mammal families. However, morphological conservatism can pose significant challenges in delineating these clades within species complexes. In this study, we leverage artificial intelligence technologies to unravel the hidden species diversity of moles (family Talpidae) in Southwest Chinas sky islands. We first employed ultraconserved elements (UCEs) to investigate the evolutionary history of talpid moles, conducted molecular species delimitation using mitochondrial and multi-locus genes, and utilized both traditional and geometric morphometrics to examine their morphological disparity. To address the challenges of morphology based cryptic species identification, we developed a deep learning Hierarchical Identification of Species NETwork (HIS-NET) to create an image-based model that analyzes four different views of the skull/mandible to distinguish genera and species hierarchically. HIS-NET not only achieved expert-level accuracy in species identification but also effectively distinguished between cryptic and known species, aiding in the identification of key morphological variation intervals. Our results support the recognition of allopatrically distributed taxa in Euroscaptor and Parascaptor as full species, thereby confirming that species diversity in this region remains underestimated. Beyond advancing our understanding of speciation in this unique and fragile ecosystem, our study serves as a proof-of-concept, demonstrating the power of deep learning in unraveling hidden biodiversity within this and other species complexes.