Cryptic Species Research Articles

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species

BackgroundMosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species.MethodsSampling was carried out in three biogeographical regions of Croatia over six years (2017–2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs.ResultsA total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna.ConclusionWe present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.Graphical

Genome divergence and reproductive incompatibility among populations of Ganaspis near brasiliensis.

During the last decade, the spotted-wing drosophila, Drosophila suzukii, has spread from eastern Asia to the Americas, Europe, and Africa. This fly attacks many species of cultivated and wild fruits with soft, thin skins, where its serrated ovipositor allows it to lay eggs in undamaged fruit. Parasitoids from the native range of D. suzukii may provide sustainable management of this polyphagous pest. Among these parasitoids, host-specificity testing has revealed a lineage of Ganaspis near brasiliensis, referred to in this paper as G1, that appears to be a cryptic species more host-specific to D. suzukii than other parasitoids. Differentiation among cryptic species is critical for introduction and subsequent evaluation of their impact on D. suzukii. Here we present results on divergence in genomic sequences and architecture and reproductive isolation between lineages of Ganaspis near brasiliensis that appear to be cryptic species. We studied five populations, two from China, two from Japan, and one from Canada, identified as the G1 versus G3 lineages based on differences in cytochrome oxidase l sequences. We assembled and annotated the genomes of these populations and analyzed divergences in sequence and genome architecture between them. We also report results from crosses to test reproductive compatibility between the G3 lineage from China and the G1 lineage from Japan. The combined results on sequence divergence, differences in genome architectures, ortholog divergence, reproductive incompatibility, differences in host ranges and microhabitat preferences, and differences in morphology show that these lineages are different species. Thus, the decision to evaluate the lineages separately and only import and introduce the more host-specific lineage to North America and Europe was appropriate.

Six in one: cryptic species and a new host record for Olixon Cameron (Rhopalosomatidae, Hymenoptera) revealed by DNA barcoding

Olixon testaceum is a widely distributed species of brachypterous parasitoid wasp (Vespoidea: Rhopalosomatidae) occurring in Meso- and South America, but little is known of its biology. Here, the first known host of O. ? testaceum is identified as the cricket Anaxipha sp. (Grylloidea: Trigonidiidae) through DNA barcoding of six Olixon larvae and their hosts. Barcoding results also indicated substantial genetic diversity within nominal O. testaceum specimens. The number of species and statistical significance of these groups were tested using Maximum Likelihood phylogenies, distance-based cluster analyses, and coalescence models. All analyses revealed at least six distinct lineages, which suggests six or more cryptic species within O. ? testaceum. Combined with what is currently known about Rhopalosoma host use, these results indicate that rhopalosomatids may be generalist rather than specialist parasitoids, and further confirm the benefits of open global collaboration and DNA barcoding in advancing taxonomic knowledge.

Unravelling male advertisement call variability in the brown tree frog (Litoria ewingii) complex by using citizen science

In the face of global biodiversity declines, conservation measures are urgently needed. However, our lack of knowledge on species’ diversity, distributions and population trends presents a major obstacle, particularly for morphologically cryptic species. Field surveys to gather this information present a logistical challenge, but the rapid rise of citizen science presents a new opportunity, generating huge volumes of data rapidly across a species’ range. We use data from a national citizen-science project to document the distribution and advertisement call variability in a group of five morphologically conserved treefrogs. Using 542 male advertisement calls across the entire range of five species, we found considerable levels of acoustic variation both among and within species. Note shape, notes per call and positions of the longest and shortest notes were most useful to delineate species. Our research will assist in species delineation and identification in the field and form the basis for a more accurate understanding of species diversity and distributions in the brown tree frog complex. We also demonstrated the utility of citizen science in documenting species distributions and diversity.

Two new species of Dictyota (Dictyotales, Phaeophyceae), D. dimorphosa sp. nov. and D. recumbens sp. nov., based on morphological and molecular investigations

ABSTRACT The brown algal genus Dictyota (Dictyotales, Phaeophyceae) is widespread, particularly in warm temperate and tropical waters, and plays a vital role in coastal ecosystems. This study reassessed the taxonomy of Dictyota, focusing on specimens from Myanmar and Japan, using a combination of morphological and molecular analyses. Molecular phylogenetic analyses using concatenated DNA sequences of rbcL, psaA, psbA, cox1 and cox3 genes revealed that Dictyota specimens from Myanmar, Japan, and other regions, belong to nine different clades, each representing a different species. Based on morphological and molecular data, seven out of these nine clades are identified as known species, while the remaining two clades are described as new species D. recumbens sp. nov. (=D. sp. A) and D. dimorphosa sp. nov. (=D. sp. B). Dictyota recumbens is characterized by the recumbent thallus, with transverse yellowish-green iridescent lines, and the formation of solitary spores irregularly distributed over the thallus. Dictyota dimorphosa is characterized by two morphological forms: decumbent and erect thalli, with bluish iridescence mainly in the decumbent thallus, and the formation of spores in oval ring shapes distributed over both thallus surfaces. Dictyota recumbens was typically found in tropical regions, while D. dimorphosa appears to be a cool-warm temperate species, distributed from northeastern Hokkaido to southern Kyushu with an extension along South Korea. Our molecular phylogenetic analyses have revealed the existence of several species whose names appeared in multiple clades, suggesting the potential for future increases in species diversity and the presence of cryptic species within certain groups.

New records of Micromegistus bakeri, Trägårdh 1948 (Acari: Mesostigmata: Parantennulidae), a mite symbiotic on carabid beetles, and notes on the species’ distribution and host specificity

Micromegistus bakeri is a purported commensal mite that spends most of its life on the ground beetle Scarites subterraneus. It is thought to sustain itself by feeding on the beetle’s dermal fluids and opportunistically scavenging on nearby organic debris, although the nature of this relationship, and whether it’s beneficial, detrimental or neutral to the host, is unclear. This species has previously been recorded in Mississippi, Texas, and Kansas. This study reports the first record of M. bakeri in Pennsylvania based on our collecting, as well as the first records for Ohio, Michigan, Missouri, Arizona, and Canada, based on previously unpublished museum records. The literature and our own survey of carabid beetles indicate that M. bakeri has a strong preference for S. subterraneus as a host and is not common on other carabids. Our own data shows a prevalence of 27% of S. subterraneus (10 out of 37) carrying M. bakeri, with an average of 3.1 (range 1–8) mites per infested beetle; none of the other 31 carabid species collected during this study carried M. bakeri. This study represents the first publicly available COI barcode sequence for this taxon. We also provide photographs of live mites and summarize suspected sightings from additional locations across North America obtained via the citizen science websites BugGuide and iNaturalist. These records indicate that M. bakeri is present throughout the known distribution of its beetle host. Suspected M. bakeri were observed on other Scarites species in North America, but their presence on these hosts needs to be confirmed via examination of physical specimens. It should be noted that the westernmost record for the species is in Arizona, based on a male specimen that exhibits 6.6% divergence in COI barcode sequence with specimens from Pennsylvania. The specimen is otherwise essentially morphologically identical to males of eastern USA, and it is therefore uncertain whether it represents a cryptic species or merely high intraspecific genetic divergence over a large geographic distance.

Hidden hotspots of amphibian biodiversity in China

Identifying and protecting hotspots of endemism and species richness is crucial for mitigating the global biodiversity crisis. However, our understanding of spatial diversity patterns is far from complete, which severely limits our ability to conserve biodiversity hotspots. Here, we report a comprehensive analysis of amphibian species diversity in China, one of the most species-rich countries on Earth. Our study combines 20 y of field surveys with new molecular analyses of 521 described species and also identifies 100 potential cryptic species. We identify 10 hotspots of amphibian diversity in China, each with exceptional species richness and endemism and with exceptional phylogenetic diversity and phylogenetic endemism (based on a new time-calibrated, species-level phylogeny for Chinese amphibians). These 10 hotspots encompass 59.6% of China's described amphibian species, 49.0% of cryptic species, and 55.6% of species endemic to China. Only four of these 10 hotspots correspond to previously recognized biodiversity hotspots. The six new hotspots include the Nanling Mountains and other mountain ranges in South China. Among the 186 species in the six new hotspots, only 9.7% are well covered by protected areas and most (88.2%) are exposed to high human impacts. Five of the six new hotspots are under very high human pressure and are in urgent need of protection. We also find that patterns of richness in cryptic species are significantly related to those in described species but are not identical.

Species delimitation in the sailfin blenny (Emblemaria pandionis) reveals cryptic endemic species diversity in the Greater Caribbean and Gulf of Mexico

Species delimitation in the sailfin blenny (Emblemaria pandionis) reveals cryptic endemic species diversity in the Greater Caribbean and Gulf of Mexico

What do We Know about Cryptic Aspergillosis?

Cryptic Aspergillus species are increasingly recognized as pathogens involved in human disease. They are ubiquitarian fungi with high tenacity in their environment and can express various resistance mechanisms, often due to exposure to antifungal agents employed in agriculture and farming. The identification of such species is increasing thanks to molecular techniques, and a better description of this type of pathogen is granted. Nevertheless, the number of species and their importance in the clinical setting still need to be well studied. Furthermore, their cross-sectional involvement in animal disease, plants, and human activities requires a multidisciplinary approach involving experts from various fields. This comprehensive review aims to provide a sharp vision of the cryptic Aspergillus species, from the importance of correct identification to the better management of the infections caused by these pathogens. The review also accentuates the importance of the One Health approach for this kind of microorganism, given the interconnection between environmental exposure and aspergillosis, embracing transversely the multidisciplinary process for managing the cryptic Aspergillus species. The paper advocates the need for improving knowledge in this little-known species, given the burden of economic and health implications related to the diffusion of these bugs.

Multiple introduction pathways of non-native Phoxinus minnows (Teleostei: Leuciscidae) in Corsica revealed by its hidden diversity and their parasites

AbstractThe introduction of freshwater fish species is a leading cause of aquatic biodiversity erosion and can spread parasites to native populations. Hidden diversity evidenced by recent taxonomic revisions can add further complexity to the issue by rendering biological assessment data incomplete. The Eurasian minnows Phoxinus are one such example of cryptic diversity, with several described species being invasive. Current non-native fish populations in the small Mediterranean island of Corsica (France) are the result of successive waves of introductions, including several Phoxinus species. This study aims at determining which Phoxinus species were introduced to Corsica using the cytochrome oxidase subunit I barcoding marker, reconstructing their introduction routes and examining their parasite communities. The study found four species in Corsica: Phoxinus phoxinus and Phoxinus csikii mainly in the northernmost studied drainage basin and Phoxinus dragarum and Phoxinus septimaniae in the Tavignano drainage basin. P. phoxinus and P. csikii were most likely introduced through a live bait wholesaler while P. dragarum and P. septimaniae were probably introduced by recreational anglers bringing their bait from continental France. The molecular study of their Gyrodactylus (Platyhelminthes: Monogenea) parasites with the ITS marker allowed us to hypothesize inter-drainage basin secondary introduction routes for P. phoxinus and P. dragarum. In several sampling sites, Phoxinus minnows had black spot disease caused by encysted metacercariae of Digenea, likely Posthodiplostomum cuticola. These parasites were also found on the brown trout Salmo trutta in a locality where this patrimonial species co-occurs with Phoxinus minnows. Barcoding should be used in fish communities monitoring to help to accurately identify cryptic species.

The cryptic microbiota of plant parasitic and entomopathogenic nematodes: diversity, effects on host biology and potential in plant protection

AbstractMany plant parasitic and entomopathogenic nematodes harbor specialized and obligate bacteria as well as viruses. Given their evolutionary persistence, such cryptic species are considered to play effective roles during their host/microbe interactions lifetime, including mutualistic, antagonistic, or yet unknown host effects. To exploit such associations in plant protection, a comprehensive view is needed linking basic evolutionary relationships to applied aspects. This requires identifying the benefit or impact that hosts, acting as pests or biocontrol agents, receive from their endosymbionts. Targeting endosymbionts that are vital for a beneficial nematode or a pest may open novel perspectives for the management of their performance and traits, such as virulence or response to plant defense reactions. Some hypotheses are proposed to develop advanced control strategies through emerging biotechnological approaches.

A review of Nearctic Lathrobium (Coleoptera, Staphylinidae), with revision and descriptions of new flightless species from the mountains of the southeastern U.S.

Species of the genus Lathrobium Gravenhorst (Coleoptera: Staphylinidae: Paederinae) from North America north of Mexico are reviewed and 41 species are recognized. Morphology and mitochondrial COI sequence data were used to guide species designations in three flightless lineages endemic to the southern Appalachian Mountains, a biologically diverse region known for cryptic diversity. Using a combination of phylogeny, algorithm-based species delimitation analyses, and genitalic morphology, five new cryptic species are described and possible biogeographic scenarios for their speciation hypothesized: L.balsamense Haberski & Caterino, sp. nov., L.camplyacra Haberski & Caterino, sp. nov., L.islae Haberski & Caterino, sp. nov., L.lividum Haberski & Caterino, sp. nov., L.smokiense Haberski & Caterino, sp. nov. Five additional species are described: L.absconditum Haberski & Caterino, sp. nov., L.hardeni Haberski & Caterino, sp. nov., L.lapidum Haberski & Caterino, sp. nov., L.solum Haberski & Caterino, sp. nov., and L.thompsonorum Haberski & Caterino, sp. nov. Two species are transferred from Lathrobium to Pseudolathra Casey: Pseudolathraparcum (LeConte, 1880), comb. nov. and Pseudolathratexana (Casey, 1905), comb. nov. Twenty-six names are reduced to synonymy. Lectotypes are designated for 47 species. Larvae are described where known, and characters of possible diagnostic value are summarized. Species diagnoses, distributions, illustrations of male and female genitalia, and a key to Lathrobium species known from the Nearctic region (including several introduced species) are provided.

A first species-wide phylogenetic analysis of small mammals from Costa Rica using mitochondrial cytochrome b

AbstractCosta Rica is within the Mesoamerican biodiversity hotspot and has about 53 native species of small mammals. This high diversity, along with recent records of new species and indications of cryptic genetic diversity, suggest that application of the DNA barcoding approach would be worthwhile. Here we used 131 tissue samples of small mammals from multiple localities in Costa Rica and sequenced the complete mitochondrial cytochrome b (1140 bp). These samples represented 17 recognized species and two taxa of uncertain status. The new sequence data were supplemented with previously published data from INSDC. Our phylogenetic analyses are consistent with and extend upon recent revisions in Heteromys, Peromyscus and Reithrodontomys and suggest possible new cryptic forms within what are currently named Melanomys chrysomelas, Nyctomys sumichrasti and Proechimys semispinosus. The previously named “Heteromys sp” is indeed likely a new species requiring a full taxonomic description. Moreover, we found new localities for previously described species substantiating recent taxonomic surveys and field guides for the small mammals of Costa Rica. To confirm the presence of cryptic species and major genetic forms in Heteromys, Peromyscus, Reithrodontomys, Melanomys, Nyctomys and Proechimys there needs to be greater sampling, additional genetic markers, morphometrics and other studies. Scotinomys also shows interesting phylogenetic subdivision, requiring further investigation.

Comparative karyotype analysis of the red brocket deer (M. americana sensu lato and M. rufa) complex: evidence of drastic chromosomal evolution and implications on speciation process.

Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

Towards a Comprehensive DNA Barcode Library of Stenochironomus Kieffer, 1919 (Diptera: Chironomidae) from China

The adoption of DNA-based assessments for biodiversity monitoring has been on the rise. However, the effectiveness of DNA-based taxonomic assignments heavily relies on the availability and reliability of DNA barcode libraries. There is growing demand for a comprehensive understanding of aquatic biodiversity and the critical role of Chironomidae, specifically Stenochironomus in freshwater ecosystems. Therefore, our objective is to develop a reference barcode library for Stenochironomus in China. From 2016 to 2021, we collected Stenochironomus specimens in diverse Chinese landscapes using malaise traps, light traps, and sweep nets. These specimens were carefully preserved for DNA extraction and barcode sequencing. Our analysis unveiled 36 unique operational taxonomic units from 180 COI barcode sequences through a Neighbor-Joining tree and Automatic Barcode Gap Discovery program, highlighting a significant diversity within the Stenochironomus species. The findings emphasize the constraints of conventional morphological identification methods, especially for species with ambiguous morphologies. It also underscores the effectiveness of DNA barcoding in revealing hidden species diversity, known as cryptic species. Consequently, this study advocates for an integrated taxonomic approach, combining morphological and molecular data, to refine species identification and conservation strategies.

Comparative Mitogenome Characterization of Cryptic Asian Seabass (Lates calcarifer) in Captivity within Peninsular Malaysia

Lates calcarifer is one of the most commercial species in the world. Yet, phylogenetic relationships among this cryptic species remain unclear despite its significance in the fishery sector. The first report on bifurcation of captive Lates calcarifer in Peninsular Malaysia (PM) inferred from complete mitochondrial genome was discussed in the present study. Two mitochondrial genomes (mitogenomes) of Lates species were sequenced and assembled. The mitogenome length was 16,627 bp and 16,515 bp for K4 and S15, respectively. The majority of PCGs in both species exhibited the common initiation marker ATG codon and ended with termination marker TAA codon. Also, the incomplete termination codon T/TA was found in both species. Most AT-skew and GC-skew values observed in the protein-coding genes (PCGs) across the two samples were negative. The phylogenetic analysis based on 13 protein-coding genes by Maximum-Likelihood tree displayed two lineages from different regions which were from Myanmar/Indian waters (K4) and Southeast Asia/ Australian waters (S15). The striking genetic distance values between both specimens indicated 13 PCGs were suffering purifying selection. This study offers significant information for future analyses of evolutionary relationships among the numerous and taxonomically puzzling perciform fishes.

Cicada minimum age tree: Cryptic speciation and exponentially increasing base substitution rates in recent geologic time

We developed a new time-calibrated tree incorporating primarily endemic along with some cryptic Ryukyu islands cicada data, following the recent publication of global cicada data by Marshall et al. (2018), Łukasik et al. (2018), Simon et al. (2019), Price et al. (2019), and Hill et al. (2021).  A total of 352 specimens were analyzed using BEAST v1. X software with a relaxed clock model. Fossil calibrations as old as Triassic were adopted largely following Johnson et al. (2018) and Moulds (2018), and a Quaternary geological event calibration was adopted following Osozawa et al. (2012, 2021b) and input into BEAST v1. X. Our timetree suggests that Tettigarctidae had a cicada basal lineage as old as 200.63 Ma, with Derotettiginae the next oldest lineage at 99.2 Ma. Tibicininae is a sister of the remaining subfamilies of Tettigomyiinae, Cicadettinae, and Cicadidae, and their species level differentiation and radiation began at 40.57 Ma. The Cicadinae clade consists of specific tribes with paraphyletic relationship, and the vicariance and adaptive radiation generated many cryptic species in each tribe. We estimated base substitution rate as a function of age, and the result strongly indicates an exponential increase of base substitution rate in recent geologic time. The consequent increase in cicada biodiversity, including generation of cryptic species in the Ryukyu Islands and surroundings, may have been driven by the generation and spreading of C4 grasses and coeval Quaternary climate change.

Circumtropical distribution and cryptic species of the meiofaunal enteropneust Meioglossus (Harrimaniidae, Hemichordata)

Hemichordata has always played a central role in evolutionary studies of Chordata due to their close phylogenetic affinity and shared morphological characteristics. Hemichordates had no meiofaunal representatives until the surprising discovery of a microscopic, paedomorphic enteropneust Meioglossus psammophilus (Harrimaniidae, Hemichordata) from the Caribbean in 2012. No additional species have been described since, questioning the broader distribution and significance of this genus. However, being less than a millimeter long and superficially resembling an early juvenile acorn worm, Meioglossus may easily be overlooked in both macrofauna and meiofauna surveys. We here present the discovery of 11 additional populations of Meioglossus from shallow subtropical and tropical coralline sands of the Caribbean Sea, Red Sea, Indian Ocean, and East China Sea. These geographically separated populations show identical morphology but differ genetically. Our phylogenetic reconstructions include four gene markers and support the monophyly of Meioglossus. Species delineation analyses revealed eight new cryptic species, which we herein describe using DNA taxonomy. This study reveals a broad circumtropical distribution, supporting the validity and ecological importance of this enigmatic meiobenthic genus. The high cryptic diversity and apparent morphological stasis of Meioglossus may exemplify a potentially common evolutionary ‘dead-end’ scenario, where groups with highly miniaturized and simplified body plan lose their ability to diversify morphologically.

Taxonomic review of the genus Hexacentrus Serville, 1831 (Orthoptera: Tettigoniidae) in Korea: An integrative study to confirm cryptic species

The genus Hexacentrus (Orthoptera: Tettigoniidae) comprises two species in Korea: Hexacentrus japonicusKarny, 1907, and H. hareyamaiFurukawa, 1941. While H. japonicus is well-known in Korea, H. hareyamai was previously confirmed as a cryptic species through a DNA barcoding study. In this research, we reviewed the Korean Hexacentrus using morphological and bioacoustic characters and conducted a phylogenetic analysis using cytochrome oxidase I (COI) for molecular identification. We describe the cryptic species H. hareyamai in Korea, which was previously recorded as H. unicolor in a prior study. Based on our findings and the biogeographical range of Hexacentrus species, we suggest that the presence of H. unicolor in Korea should be excluded. However, since a systematic comparison of Hexacentrus species, including H. unicolor, in Southeast Asia has not been investigated in detail, further studies are needed.

Comprehensive morphological and phylogenetic inferences of star-shaped fungus Astraeus (Diplocystidiaceae) from sal-dominant tropical and subtropical Pinus-Shorea forests in India: an integrative taxonomic analysis

Classifying fungus based on morphological traits is an effective strategy to distinguish between puffballs, earthballs, earthstars, and other gasteroid fungi, as well as for identifying sub-groups of closely related taxon. However, to delimitate taxa it should be addressed with caution, since cryptic species have been described recently in different genera of gasteroid fungi. Astraeus is a star-shaped fungus that has piqued the interest of mycologists worldwide, including India. These endearing fungi have tremendous nutritional and therapeutic benefits, but their molecular and phylogenetic placement in India is uncertain. An integrative taxonomic approach was used to identify and resolve ambiguities within the genus. This study, the first to elucidate the distribution, taxonomy, and phylogeny of the genus Astraeus in the mixed subtropical Pinus-Shorea forest and tropical dry deciduous Shorea dominated forest in India, aims to shed light on these indispensable ectomycorrhizal fungi. The phylogenetic analysis assigned all thirteen ITS DNA barcoding Indian sequences of Astraeus to the Southeast Asian clade, reinforcing the genus Southeast Asian origin. Detailed species descriptions, line diagrams, SEM images of basidiospores, ITS nrDNA based phylogeny, and a dichotomous key are provided. Mycoobservations of the taxon from Pinus-Shorea subtropical and Shorea-dominated forests were additionally reviewed.