Integrative taxonomy of the genus Coridius Illiger, 1807 (Hemiptera: Heteroptera: Dinidoridae) reveals hidden diversity and three new species from North-East India

The genus Coridius Illiger, 1807 (Heteroptera: Dinidoridae) comprises a group of phytophagous terrestrial bugs consisting of 36 species distributed in the Afrotropical and Indo-Malayan regions. In several communities in northeastern India, insects are recognised as a delicacy, medicine, and a nutritional supplement, with Coridius being a popular delicacy. However, Coridius has received little taxonomic attention to date due to large intraspecific variations, inadequate taxonomic treatments, and the rarity of many species. To address this gap, an integrative taxonomy of the genus was performed. Two mitochondrial genes, viz., cytochrome oxidase subunit 1 (COI) and 16S rRNA, were sequenced to reconstruct the phylogenetic relationships within Coridius. We performed both maximum likelihood (ML) and Bayesian inference (BI) to develop a species tree, followed by the Bayesian implementation of the Poisson tree process (bPTP) and Assemble Species by Automatic Partitioning (ASAP) as an additional test to assess species boundaries and delimit operational taxonomic units. A linear discriminant analysis (LDA) of four key morphological characters was then performed to identify species groups. Overall, our analysis supported the establishment of three new species: Coridius adii sp. nov., Coridius esculentus sp. nov., and Coridius insperatus sp. nov., and revealed six distinct lineages within Coridius chinensis (Dallas, 1851). Linear discriminant analysis of morphological characters indicated the clustering of eight species. The species status of Coridius nigriventris (Westwood, 1837) stat. rev, formerly synonymized under Coridius nepalensis (Westwood, 1837), is reinstated in this study. Further, we revised the genus Coridius from India and rediscovered Coridius assamensis (Distant, 1902) and Coridius fuscus (Westwood, 1837) after 100 years.


Introduction
Species serve as the fundamental units of biodiversity [1], and reliable species delimitation and classification can enhance inferences in biosystematics and various other scientific fields, including biogeography, epidemiology, and conservation biology [2,3].However, accurate species delimitation often poses several conceptual and empirical challenges to biologists, which are further augmented while dealing with i) cryptic species [4], ii) lineages exhibiting shallow divergence [5], and iii) taxonomically complex systems [6].Consequently, setting criteria for species delimitation has invoked a series of debates among systematic biologists in recent decades [7,8].A well-established species delineation criterion should be repeatable, provide independent assessments of morphologically defined lineages, and uncover previously unknown species [9].Monophyly is one accepted criterion for supporting separate species [10].However, taxonomic issues may arise when molecular phylogenies and morphological classifications are incongruent.In such cases, combining information from multiple yet conventionally disparate datasets and methodological approaches has proven useful in delineating species boundaries with better accuracy [11,12].
Integrative taxonomy, which combines diverse datasets such as morphological, ecological, and molecular data (nuclear and mitochondrial DNA), has gained significant attention among biologists [13,14].This approach serves as a powerful tool for delineating species boundaries and offers a practical solution to the "taxonomic impediment," a major obstacle in biodiversity research [15][16][17].It allows researchers to harness the combined strength of various data types, including genetics, morphology, biochemical, and ecological information, to more accurately infer species boundaries [3,18].This approach is particularly relevant when inferences based on morphology fail, especially when dealing with lineages that contain species complexes or cryptic species [19,20].Thus, the integration of multiple datasets allows a more comprehensive understanding of the complex evolutionary processes that give rise to unrecognized species [21], such as ecological speciation [22] and incomplete lineage sorting [5].This approach moves taxonomy beyond species descriptions and provides a more nuanced understanding of phylogenetic relationships within and between taxa.
The genus Coridius belonging to the family Dinidoridae is distributed throughout the Afrotropical (22 species), Australasian (1 species), Indomalayan (16 species), and Palearctic (2 species) realms, with 9 species known from India [23,24].These are phytophagous insects, primarily feeding on plants of the Arecaceae, Cucurbitaceae, Fabaceae, and Moraceae [25].Although the host plants of many Coridius species are known [25], the life cycle and ecology of most species remain underexplored.
The genus Coridius is economically important, as species of this genus are widely consumed by the native people of northeast India.Members of this genus are also known for their various therapeutic properties [26].These insects are collected from the dry riverbeds (Fig 1A and 1B), where they diapause during the winter, and are sold in local markets.However, the impact of this widespread consumption of Coridius on its population is not very well understood, and a lack of baseline information on the ecology and evolution of this group further hinders the development of any bioprospecting and conservation strategies.
Until 1980, a total of 37 species of Coridius were described [27] based on their morphological characteristics, including traits such as coloration and male genitalia.However, several of these species were later synonymized or reassigned to other genera.Due to the intricate taxonomic complexities in Coridius, identifying species boundaries based solely on morphological and anatomical characteristics may be inadequate and could lead to further confusion.Moreover, Coridius species show a range of host preferences and cryptic morphological differences, making it an ideal study system to apply integrative taxonomic methods and reassess species boundaries.
In this context, to address the taxonomic complexities within the genus Coridius, we have set three primary objectives.Firstly, we aim to conduct a comprehensive systematic revision of Coridius species found in India, focusing on the highly diverse northeast region with improved sampling.Secondly, we intend to identify key morphological characters to verify previous systematic treatments of the genus.Lastly, we attempt to develop a multilocus phylogeny and employ species delimitation analysis based on molecular data as an additional test to gain confidence in the recognized species groups.

Taxon sampling
We collected 150 specimens from October 2018 to January 2021 from 21 different locations across India (Fig 1C and 1D).The collected specimens were preserved in absolute alcohol for morphological and molecular studies.Nomenclatural acts.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article.This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN.The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/".The LSID for this publication is: urn: lsid:zoobank.org:pub:3BCC7D9C-FC18-4A74-B923-4900C8CDAD41.
Biodiversity board permits.For this study, all required permits were obtained from the respective biodiversity boards in the respective regions.In Arunachal Pradesh, permit number SFRI/APBB/9/2011/4019, issued by the Arunachal Pradesh Biodiversity Board.In Nagaland, permit number NSBB/31/PABR/2016/652, granted by the Nagaland State Biodiversity Board.In Manipur, the necessary permissions were obtained from the Manipur Biodiversity Board, permit number 3/31/2013-17/APCCF/BIO & NTFP: 67.

Morphological analysis
The collected specimens were mounted and studied under Zeiss Stemi 305.Photographs and measurements of the specimens were taken using Keyence VHX6000 and processed using Adobe Photoshop 2024.The last two abdominal segments were detached and boiled with 10% KOH for 10 minutes to separate the pygophore.The pygophore was then dissected to separate the parameres.The dissected specimen was briefly rinsed with dilute acetic acid and distilled water, followed by 70% alcohol, and then mounted again.
Measurements for all species are given in the S1 File.
We measured the head length (HL) and head width to the eye (HW) to calculate the HL/ HW ratio; similarly, we measured the length of pronotum (PL) and maximum width of pronotum (PB) to calculate the PL/PB ratio; length of scutellum (SL) and maximum width of scutellum (SB) to calculate the SL/SB ratio; maximum breadth of the body (MB); and total body length (TBL).Linear Discriminant Analysis (LDA) was performed using the morphological characters of 10 species.Two species (C.adii sp.nov., and C. assamensis) were dropped from LDA due to insufficient samples.The LDA was performed using R v. 4.1.3 packages 'klaR' [29], 'psych' [30], 'MASS' [31], and 'ggord' [32].

DNA extraction and sequencing
Total genomic DNA was extracted from sternal muscles using the QIAGEN DNeasy blood and tissue kit following the manufacturer's protocol.We PCR amplified and sequenced two mitochondrial loci, Cytochrome C Oxidase Subunit 1 (COI F: 5' GGTCAACAAATCATAAAG ATATTGG 3', COI R: 5' AAACTTCAGGGTGACCAAAAAATCA 3') [33] and 16S rRNA subunit (16s F: 5' CGCCTGTTTATCAAAAACAT 3', and 16S R: 5' CCGGTCTGAACTCA GATCACGT 3') [34].The PCR conditions for amplifying the COI consisted of an initial denaturation at 95˚C for 5.00 min, followed by 40 cycles at 94˚C for 30 s, 46˚C for 1.00 min, and 72˚C for 1.45 min, and a final extension at 72˚C for 15.00 min.Similarly, for the 16S initial denaturation, the temperature was set at 95˚C for 5.00 min, followed by 40 cycles of denaturation at 95˚C for 30 sec, an annealing temperature at 53˚C for 1.00 min, and 72˚C for 1.30 min, followed by a final extension at 72˚C for 10 min.We could not sequence any nuclear genes due to the lack of a comprehensive dataset available in GenBank.All PCR products were purified and sequenced in the Barcode Biosciences laboratory (Bengaluru, India).The resulting DNA sequences were deposited in GenBank (see Table 1).

Sequence alignments
Sequences were obtained from 22 specimens consisting of eight species (    Lygaeoidea and treated them as outgroups from the GenBank.The sequenced chromatograms were manually inspected for quality and aligned using the software program Geneious Version 2023.1.1.The gaps in sequences were treated as "missing".

Phylogenetic analysis
The phylogenetic analysis was conducted based on a total of 61 sequences from 11 species of Coridius.The final combined alignment consists of 1002 nucleotides, of which 248 sites were parsimony informative.The best-fit nucleotide substitution model for Bayesian inference was selected for both genes based on the Bayesian information criterion (BIC) in PartitionFinder 2.0 [35] (Table 2).The HKY+I+G substitution model was assigned to the first codon position, and the TRN+G model was assigned to the second and third codon positions for COI.The HKY+I+G substitution model was assigned for the first, second, and third codon positions in 16s.We ran maximum-likelihood (ML) analyses in IQ-Tree Web Serve [36] with 1000 bootstrap replicates under the GTRGAMMA substitution model in the combined dataset.A Bayesian inference (BI) was performed in the software program MrBayes 3.2.1 [37].Four independent chains of Markov chain Monte Carlo (MCMC) were performed for 50 million generations with a sampling frequency of 500.The convergence of MCMC chains was assessed in Tracer [38].The first 25% of the runs were discarded, and the remaining trees were used to build a consensus tree and estimate Bayesian posterior probabilities (PP).We used FigTree 1.4.4 [39] to visualize and edit the phylogenetic trees.Species delimitation.Molecular species delimitation analyses were conducted using the Bayesian Poisson Tree Processes (bPTP) method through the online web server available at http://species.h-its.org/.The analyses consisted of 100,000 Markov Chain Monte Carlo (MCMC) generations with a thinning parameter set to 100 and a post-burn-in value of 0.1.Additionally, a single-rate PTP analysis was performed using the ML tree generated from IQTREE (https://mptp.h-its.org/#/tree),with the default p-value settings [40].To further support our species delimitation method, we employed the Assemble Species by Automatic Partitioning (ASAP) analysis, utilizing the genetic distance matrix method [41].This analysis was conducted through a web-based interface accessible at https://bioinfo.mnhn.fr/abi/public/asap/, after setting the default parameters.For this analysis, we selected the Kimura (K80) (TS/ TV) 2.0 model.The best two ASAP scores resulting from this analysis have been referred to as ASAP 1 and ASAP 2.

Phylogenetic inference
Maximum Likelihood (ML) and Bayesian Inference (BI) tree analyses of two concatenated genes (COI and 16S) showed similar topologies with high support (maximum likelihood bootstrap support, MLBS) values and posterior probabilities (PP) (Fig 4).Lygaeidae was used as the root for the tree.The family-level relationship showed Urostylididae (PP: 1, MLBS: 95) as the sister taxa to all other Pentatomoidea in both analyses.Also, we found clear evidence of  at least six clades.Interestingly, C. esculentus sp.nov.was found to be grouped within a clade alongside C. chinensis but formed a distinct branch (PP: 0.82, MLBS: 52).This outcome was in accordance with our expectations, as C. chinensis and C. esculentus sp.nov.have similar coloration but differ significantly in anatomy (see the discussion section and Fig 4).Furthermore, our investigation revealed that the African species C. viduatus and C. ianus form a sister group (PP: 1, MLBS: 100), aligning with our expectations due to their striking morphological similarity (Fig 4).
Species delimitation and new lineages.The estimated molecular operational taxonomic units (MOTUs) were not consistent among the methods: 13 for PTP, bPTP for 14, ASAP 1 for 14, and ASAP 2 for 25 (excluding out-groups) (Fig 4).The species status of C. insperatus sp.nov.was supported by the analysis.Coridius nepalensis and all undetermined species were treated as a single species in the analysis.In the analysis, PTP, bPTP, and ASAP 1 suggested grouping four species, namely C. singhalanus, C. adii sp.nov., C. assamensis, and C. chinensis, as a single species, whereas ASAP 2 proposed each of these species should be considered distinct species.Similarly, C. esculentus sp.nov. is found nested with four other individuals of C. chinensis, forming a well-supported clade in the phylogenetic analysis (PP: 0.99, MLBS: 95).Both ASAP 1 and ASAP 2 have recognized them as distinct species.Coridius nigriventris (JQ387600) clustered with C. ianus (KU043393) in ML and BI topologies, while the PTP and bPTP analyses suggest these to be two separate species.
Interestingly, our analysis showed that C. chinensis belongs to a taxonomic species complex forming six different lineages.The first lineage was composed of a single specimen found in Arunachal Pradesh.The second lineage included two specimens from Guizhou Province, China (KJ603304 and KJ603305) and one from Manipur, India.The third lineage consisted of a single specimen found in Nagaland.The fourth lineage was formed from two specimens from Manipur and three from China (JF700134, JF700135 and JF700136) with strong node support (PP: 0.99, MLBS: 98).The fifth lineage included three specimens from Nagaland (C.chinensis 4; C. chinensis 3; and C. chinensis 5) and C. chinensis (JQ387599) from China.The sixth lineage consisted of a single specimen from Kerala, India, C. chinensis (KU043392).
Diagnosis.Head small, triangular, broader than long, mandibular plates longer than clypeus, rounded, and meeting or not in front of clypeus.Ocelli prominent, placed closer to eyes than to each other.Eyes globular, pedunculate.Labium four segmented, reaching about middle of meso-sternum, first and second segment mostly sub-equal, third and fourth smaller and sub-equal.Bucullae prominent, raised above the level of labium.Antennae four or five segmented, first segment surpassing apex of head, second and third subequal or sometimes second longer or shorter than third, fourth generally long with setae on both sides, fifth cylindrical, and finely setose.In some species second, third and fourth antennal segments grooved and flattened.
Pronotum declivous, trapezoidal.Lateral margins straight or rounded, sometimes slightly raised or carinate; dorsally rugulose punctate.Calli rugulose and smooth.Anterior margin concave behind head.Anterior angles slightly broader than width at eye.Humeral angles rounded, slightly truncate.Posterior margin moderately straight over scutellum.
Scutellum broader than long, longer than clavus, lateral margin distinctly sinuate, and its apical region broadly rounded.
Hemelytra longer than abdomen.Clavus narrow, short, corium broad.Membrane long and broad with numerous longitudinal veins.
Pro-sternum deeply sulcate mesially, its lateral area distinctly raised or flat, posterior margin slightly sinuate in lateral region.Pro-coxal area distinctly raised, pro-coxae very close together.Meso-sternum smooth on disk with median groove and longitudinal rugae.Metasternum very narrow between coxae.Metathoracic scent gland situated close to metacoxae, more ventral in position with narrow peritreme and well-developed evaporatoria.
Coxae close together in a linear series.Femora slightly curved or straight with spines underneath in distal half, tibiae distinctly sulcate and with spines distally.First segment of tarsus more setose, second and third with very sparse, long setae.Claws divergent, pulvilli distinct, well developed.Hind tibia in females slightly dilated and with oval tympanal organ.
Abdomen broad, connexivum moderately or well exposed.Segmental sutures sinuate, seventh sternum longest.Spiracles black, situated closer to anterior margin than lateral margin of segment.Spiracle of first visible abdominal segment partially or totally covered by metasepimeron.Trichobothria distributed in pairs posterior to pseudosutures and spiracle.
Pygophore visible from dorsal as well as from ventral sides, basally convex, flat in distal half, ventral surface rugulose; ventral rim rounded or notched medially or sometimes raised or concave or straight; dorsal rim moderately concave or straight.Parameres short or slender, apical tip rounded or blunt or pointed.
Etymology.The species name is the Latin adjective insperatus (meaning 'unexpected' in Latin) because it is an unexpected member of the genus Coridius with four segmented antennae.Diagnosis.Overall colour yellowish brown.Pronotum ventrally pale yellowish brown, lateral margins black.Legs yellowish brown, femora slightly darker.Antennae with first segment basally ochraceous, distally dark brown, second and third black, fourth ochraceous except a minute region at base and apex which are black.Pronotum with a levigate pale line from Description.Overall structure, Body finely punctate; pronotum with a few transverse rugae in posterior two thirds; region around calli smooth, shining, with sparse punctures.Scutellum rugulose punctate in basal two third, apical region granulated.Prosternum with a distinct elevated smooth region on either side of median groove, laterally rugulose and coarsely punctate.Meso-and meta-sterna smooth medially, rugulose and coarsely punctate in pleural regions.Body nearly smooth medially on abdominal sternites, very finely punctate laterally.Legs laterally compressed; tibiae distinctly sulcate and spinose dorsally and ventrally, more compressed than femora; femora with ventral spines; first segment of tarsus densely setose, second and third with very sparse, long setae; claws widely separated, dark brown to black in distal half; pulvilli distinct, well developed (Figs 5A and 6A).
Head transverse, two times wider than longer at eyes, mandibular plates much longer than clypeus, parallel sided, rounded anteriorly and reaching in front of clypeus (Fig 9A).Dorsal surface of mandibular plates with irregular folds and scattered punctures.Vertex coarsely punctate medially, with longitudinal rugae and grooves near eyes.Eyes large, globular and pedunculate.Ocelli well developed, slightly elevated.Interocellar distance 1.51 times distance between eyes and ocelli.Antennae four segmented, first segment cylindrical with few setae, surpassing apex of head, second and third distinctly laterally compressed, second segment with many setae and longitudinal grooves in basal one fourth with fine spine like setae on the sides, third segment finely rugulose, with spine-like setae on both sides, slightly dilated in basal half while fourth segment rounded, gradually tapering, finely punctate and covered with numerous small and sparse, long setae.
Labium four segmented, reaching middle of meso-sternum; first and second segments subequal, third (0.81 mm) and fourth (0.64 mm) smaller.Bucculae prominent, raised above the level of labium.
Pronotum rugulose punctate, calli less punctate, somewhat shining.Anterior margin of pronotum concave behind head, slightly truncate medially behind eyes.Width at anterior angles of pronotum only slightly wider than width at eyes.Lateral margins straight, slightly raised.Humeral angles rounded, posterior margin straight.Prosternum deeply sulcate in middle, its sides distinctly raised.Posterior margin of pro-sternum slightly sinuate in lateral parts.Pro-coxal area distinctly raised, pleural area coarsely punctate and rugulose, closer to coxae.Mesosternum smooth medially with shallow groove in anterior three fourth and slightly deeper groove in distal one fourth, especially near meso coxae.Pleural area coarsely punctate and rugulose.Metasternum very narrow between coxae, metasternum not covering spiracle of first visible abdominal segment posteriorly, leaving it partly exposed.
Scutellum triangular, median region elevated, lateral margins distinctly sinuate in the middle, apical region broadly rounded.Apical area finely punctate and rugulose, with coarse punctures.Median levigate line in basal half continuing pronotal levigate line.
Hemelytra.Clavus narrow, short, corium very broad, very finely and closely punctate and rugulose, punctures at margins slightly coarse.Membrane long and broad with numerous longitudinal veins and three basal cells.Hemelytra longer than abdomen.Extreme basal region of corium upturned forming shallow groove, its margin black, similar to that of pronotum, remaining margin of corium flat.
Abdomen broad, with sinuate intersegmental sutures sinuate, seventh sternum longest.Spiracles situated closer to anterior margin than to lateral margin of segment.Trichobothria distinct, a pair situated posteriorly to pseudosuture, which is posterior to spiracle.
Male genitalia.Pygophore 0.91 times wider than long, somewhat rectangular, and sclerotized.Basally convex, flat in distal half, ventral surface rugulose and ventral margin with fine black denticulation, with sparse setae.Ventral rim with rugae and numerous long ochraceous Geographical distribution.Presently known only from Arunachal Pradesh, India.
Remarks.Coridius insperatus sp.nov., is a very distinct species compared to other congeneric species.It can be easily distinguished by its four segmented antennae and cupreous dorsal colouration, while all other species in the genus are dark brown, yellow to black dorsally, and have five segmented antennae.In the family Dinidoridae, the genus Amberiana Distant, 1911, has one species with four segmented antennae (A.major Schouteden, 1912), while other species (A.montana Distant, 1911) have five segmented antennae [23,24].This species is edible and consumed by the Nyishi and Adi tribes of Arunachal Pradesh.In addition, we found that this species is sold in the Nirjuli local market (Dist.Itanagar, Arunachal Pradesh) during the winter season (October-January).
Etymology.This species is dedicated to the Adi tribe, one of the populous groups of Arunachal Pradesh, inhabiting mainly along the Siang valley, who consume this species for food.
Diagnosis.Overall pale brown to dark brown with irregular patches of yellow on pronotum, and corium.Lateral margins of pronotum black, pronotum with a levigate pale line from posterior region of calli to mid-point of its length.First four segments of antennae black, minute regions at base and apex are ochraceous, fifth ochraceous except a minute region at base and apex which are brown (Figs 5B and 6B).Connexival segments with yellow spot in between segmentation; ventrally, dark brown.Labium pale brown.Pleural area and area near spiracles dark brown or blackish brown.All legs yellowish brown, femora slightly darker, claws dark brown to black in distal half.
Description.Overall structure.Body finely punctate, region around calli smooth, shining; scutellum punctate rugulose, its apical region somewhat rounded.Prosternum with smooth flat region on either side of the median groove, laterally rugulose and coarsely punctate.Meso and metasternum smooth.
Head transverse, 1.92 times wider than long, mandibular plates longer than clypeus, rounded anteriorly and meeting in front of clypeus (Fig 9B ), dorsally with irregular folds and scattered punctures and very sparse golden setae.Eyes globular and pedunculate.Ocelli prominent, interocellar distance 2.16 times distance between eyes and ocelli.Antennae five segmented, first segment (0.87 mm) cylindrical with sparse setae surpassing apex of head, second (1.59 mm) and third (1.64 mm) subequal, compressed; second, third and fourth segments with longitudinal grooves and fine spine-like setae on the sides, fourth segment slightly dilated in basal half, fifth finely covered with numerous small and sparse long setae, pointed at the tip.
Pronotum declivous, rugulose, punctate, somewhat shining.Anterior margin of pronotum concave behind head.Lateral margins almost straight, slightly raised.Humeral angles rounded, posterior margin of pronotum straight.Prosternum sulcate in the middle, its lateral area flat.Meso and metasternum smooth on disk with a shallow, rugulose median groove.
Hemelytra shorter than abdomen, clavus narrow, short, corium broad, finely, and closely punctate, rugulose.Membrane long and broad with numerous longitudinal veins.Legs laterally compressed; femora with spines ventrally, tibiae distinctly sulcate and spined ventrally as well as dorsally; first segment of tarsus densely setose, second and third with very sparse long setae; claws widely separated, pulvilli distinct, well developed.
Abdomen broad, seventh sternum longest.Spiracles black, closer to anterior margin than lateral margin of respective segment.Trichobothria distinct with a pair situated behind the pseudosuture, posterior to spiracle.
Male genitalia.Pygophore longer than broad, sclerotized, basally convex, flat in distal half.Ventral rim slightly notched, irregular and without setae ( Geographical distribution.India.This species is known only from its type locality (Pasighat: Arunachal Pradesh).
Etymology.The species name 'esculentus' is the Latin adjective for edible, denoting its delicacy.
Diagnosis.Dorsally bronzy black, antennae black, except fifth segment which is ochraceous with black base.Legs dark brown, mid-and hindcoxae are pale brown.Lateral margins of connexivum black.Ventrally rostrum, pleural area and last two segments of abdomen are blackish brown.Mid-and hind-coxae pale brown.Second to fourth abdominal segments brown (Figs 5C and 6C).
Description.Head transverse, 1.32 times wider than longer.Lateral margins in front of eye distinctly sinuate so that apex of head looks rounded (Fig 9C).Mandibular plates longer than clypeus, coarsely punctate.Clypeus transversely ridged.Eyes moderately large, pedunculate.Ocelli prominently closer to eyes than to each other; interocellar distance 1.61 times distance between eyes and ocelli.Antennae with scattered black setae especially on second to fourth segments and a few on fifth.First segment slightly surpassing apex of head, rounded, second, third and fourth laterally compressed, flattened with medial depression; fourth longest, slightly dilated beyond middle, fifth segment fusiform.Labium four segmented, first segment reaching base of head, second segment longest reaching base of fore coxae, third and fourth segment equal in length with fine setae.
Scutellum punctate, apex broadly rounded.Hemelytra.Clavus broad at base, narrowed distally and shorter than scutellum; corium broad, finely rugulose punctate.Membrane broad, with a series of basal cells and many parallel longitudinal veins.Hemelytra surpasses abdominal apex.
Legs.Femora laterally compressed.Tibiae sulcate with numerous spines.Tarsi well developed, first and third segments equal with long setae, middle shortest; first segment with dense setae underneath, claws and pulvilli well developed; claws divergent.
Abdomen very finely rugulose, though appear smooth, slightly punctate medially.Seventh sternum longest, posterior third rugulose, covering convex ventral portion of pygophore.Exposed portion of pygophore finely rugulose.Spiracles prominent.Trichobothria as usual for the genus with a pair posterior to spiracles.
Geographical distribution.Presently known only from Arunachal Pradesh, India.
Remarks.This species is a delicacy among the ethnic communities.Consumption in large quantities can be neurotoxic, and the person becomes photophobic, such as wanting to hide under the carpet or cot; or, as the people believe, 'they begin to act like the bug, which hides under the stones or goes into cracks.If medical treatment is not sought, this behaviour can remain for a longer period, like for many months or even longer (personal communication with native people).The chemicals secreted by the bugs through the metathoracic glands might be the reason for intoxication.
Head wider than long, mandibular plates longer than clypeus, rounded anteriorly and meeting in front of clypeus (Fig 9J).Eyes globular, pedunculate.Ocelli well developed, closer to the eyes than to each other; interocellar distance 1.75 times distance of between eyes and ocelli (Fig 9J).Labium four segmented, reaching anterior margin of meso coxae.First segment extending beyond head.
Remarks.This species was described from Laos and is known only from its type locality.Lis [24] mentioned that this species is present in India without any further information.In their world catalogue, Rolston et. al.,[42] also mentioned China and Vietnam.Later, Kocorek [43] reported this species for the first time from Thailand.The specimens used here were donated by Marcos Roca-Cusachs, which were collected from the Laos and are identical to the holotype, as observed from images (Fig 7A and 7F).description.Durai synonymized this species with C. nepalensis in 1987, considering the similarity of the aedeagus with the latter.However, in this study, we used the sequence deposited as C. nigriventris (accession #JQ387600) in GenBank for molecular analysis.We found that C. nigriventris and C. nepalensis form separate lineages (see Fig 4).Additionally, upon comparing type images, we noted some morphological differences.Therefore, we reinstate the status of C. nigriventris as a valid species.
Head two times wider at eyes than longer, equal to the width of anterior pronotal margin.Mandibular plates smooth, much longer than clypeus and meeting in front of it, lateral margins sinuate, and head rounded at apex; clypeus faintly ridged mesially (Fig 9D).Eyes moderately large, pedunculate.Ocelli prominent, rounded, interocellar distance 1.97 times distance between eyes and ocelli.Antennae five segmented, all segments with fine sparse setae, fifth segment pilose.First segment surpassing apex of head, second segment 0.71 times shorter than third, fourth segment longest, fifth segment fusiform in shape.Labium four segmented, reaching to mid coxa; first segment reaching to base of head, second longest, third and fourth together equal to second.
Male genitalia.Exposed region of pygophore declivous basally, flat in distal half and with very sparse long setae.Elongate, somewhat squarish and sclerotized; ventral rim not rounded and with numerous long brown setae (Fig 10D ); ventrally convex, punctate and without setae (Fig 11D).Parameres short, sclerotized, medially rounded, and broad and apically narrowing; outer margin irregular and appears denticulate and setose apically; inner margin sometimes a little concave (Fig 12D).
Remarks: Presence of this species in India was mentioned by Lis [24] without giving any illustrations.
Diagnosis.Overall body yellowish brown to dark brown; pale yellow ventrally, brown in areas near coxae and sternum rest dark brown.Lateral margins of pronotum not black.Antennae with first segment basally ochraceous, distally dark brown, second and third black, fourth ochraceous except a minute region at base and apex which are black (Figs 5F and 6F).Legs brown to blackish brown, tibia slightly darker.
Head rounded at apex, with mandibular plates much longer than clypeus (Fig 9F ); eyes large, bulbous, silvery in colour.Ocelli yellowish brown placed closer to eye than to each other.Labium four segmented reaching middle of mesosternum.Antennae five segmented, first segment reaching apex of head, fifth segment blackish brown.
Pronotum glossy and finely punctate, anterior border deeply concave, lateral margins curved, posterior margin straight; calli smooth, shining with sparse punctures.Pro-sternum with a distinct elevated smooth region on either side of the median groove, laterally rugulose and coarsely punctate.Meso and meta sternum smooth medially, rugulose and coarsely punctate in pleural regions.Femora with spines and setae underneath, tibae distinctly sulcate and spined ventrally as well as dorsally, first segment of tarsus densely setose, second and third with very sparse long setae; claws widely separated, pulvilli distinct, well developed.
Scutellum long, rugulose punctate in basal two third; reaching half-length of abdomen, apex rounded.
Redescription.Head 1.86 times wider than longer, deeply punctate, mandibular plates almost two times longer than clypeus and meeting in front of it; apex of head rounded, lateral margin deeply sinuated (Fig 9G), lateral border of mandibular plates and area in front of ocelli smooth.Eyes, large, pedunculate.Ocelli rounded; interocular distance almost twice distance between ocelli and eyes.Antennae five segmented, first segment passing apex of head, second segment 0.70 times longer than third, fourth segment longest, fifth segment 0.44 times smaller than fourth.All segments with sparsely distributed setae, second, third and fourth segments compressed, fifth fusiform.
Pronotum declivous, trapezoid, with densely packed deep punctures, lateral margins smooth and raised as carina; anterior margin concave behind head, anterior angles blunt; posterior angles obtuse, posterior margin of pronotum more or less straight; calli prominent, smooth and without punctures.
Legs.Coxae rounded; femora slightly inwardly curved, laterally compressed with a series of small stout spinules and sparse long setae; tibiae almost straight and with numerous series of spines on both sides; tarsus with long golden pubescence underneath, pretarsus with few long setae; claws well developed and divergent.
Abdomen very finely rugulose, median region slightly less rugulose and smooth, seventh sternum longest, rugulose in posterior third, setose at posterior border; Connexivum exposed with pale yellow patch in between segmentation.Spiracles rounded, prominent, closer to anterior border than to lateral border of respective segment.Trichobothria paired, posterior to spiracles.
Remarks: C. assamensis is often collected from the riverbeds in Dibang valley and consumed by the indigenous communities especially by the Nyishi tribe.
Redescription.Head transverse, 0.41 times wider than longer, almost of same width as anterior pronotal angles; lateral margins of head in front of eyes distinctly sinuate, so that head apex looks rounded at apex.Mandibular plates longer than clypeus, meeting in front of it (Fig 9K ), transversely rugulose, coarsely punctate.Clypeus transversely ridged.Eyes moderately https://doi.org/10.1371/journal.pone.0298176.g014large, pedunculate.Ocelli are prominently closer to eyes than to each other; area between eyes and ocelli coarsely punctate on the disk, slightly less coarsely punctate in front of ocelli.Antennae with scattered black setae especially on second to fourth segments, first segment slightly surpassing apex of head, cylindrical, second and third flattened with fine rugae, fourth also flattened and slightly dilated beyond middle, fifth ochraceous and densely setose with dense, short and sparse, long setae.Labium four segmented, first segment reaching base of head, fourth segment reaching about middle of mesosternum.
Prosternum coarsely punctate medially, pleura finely and rugulosely punctate.Meso-sternum medially sulcate with two broad shining smooth patches on either side of midline, lateral area rugulose with scattered punctures.Metasternum narrow with a medial sulcus between meta coxae.
Hemelytra.Clavus broad at base, narrowed distally and shorter than scutellum; corium broad, finely rugulose punctate.Membrane broad, with a series of basal cells and many parallel longitudinal veins.Hemelytra surpasses tip of abdomen.
Femora laterally compressed, punctate, ventrally with spinules.Tibiae sulcate with more prominent and numerous spines.Tarsi well developed, first and third segment sub-equal, middle shortest; first segment with dense mat of setae underneath, claws and pulvulli well developed; claws divergent.
Abdomen slightly wider in front of middle of its length, slightly thinner than humeral angles.Abdomen very finely punctate and rugulose, less punctate medially and appears smooth.Seventh sternum longest, posterior third rugulose, setose at posterior border, covering convex ventral portion of pygophore.Eighth sternum not visible externally in males.Spiracles prominent, situated closer to anterior border than to lateral margins of segments.Trichobothria posterior to spiracles.
Male genitalia.Exposed portion of pygophore finely rugulose, punctate ventrally with sparse long brown setae, margin finely denticulate.Anterior third of pygophore ochraceous, posterior two thirds more sclerotized, black.Ventral rim of the pygophore triangular, rounded or medially raised and straight, denticulate and without long setae (Fig 15).Ventrally moderately convex, with punctures and without setae (Fig 16).Parameres highly sclerotized with rugae, medially broad and rounded and apically narrowing, outer margin serrated and setose apically, inner margin straight (Fig 17) and with lobe basally.
Remarks.The umami taste of C. chinensis makes it a popular dish among ethnic tribes in Northeast India.However, consuming this bug can cause problems like dizziness, nausea, and vomiting in some people.It can lead to a loss of strength and a condition of semi-consciousness in certain people (Boyane, personal communication with native people).
Different morphs in C. chinensis .
Remarks: Eight samples of Coridius chinensis (Dallas, 1851) formed six lineages in our analysis.Although all samples are keyed out to Coridius chinensis, they show slight variations in the body color and structure of pygophores and parameres.We studied the paralectotype of C. chinensis and found that the basal ¼ of the fifth antennal segment is black in all specimens.C. chinensis 3-5 have yellow ocelli, while all other have black ocelli.These three samples, which share similar morphology, were collected from the same state.

Phylogenetic consideration
In our analyses, we observed robust family-level relationships within the Pentatomoidea superfamily.Specifically, we found that Dinidoridae formed a monophyletic group and sister taxa to Tessaratomidae.The previous studies showed Dinidoridae does not form a sister relationship with Tessaratomidae [45], whereas our results, as well as a few studies [46,47], supported the sister relationship between these taxa.
In the integrated framework, our findings from molecular and morphological studies revealed a high convergence in the species delimitation of most Coridius species.Tree-based delimitation PTP and bPTP retrieved 13 and 14 lineages, respectively, including three new species.Except for C. chinensis, which formed six putative species.Similarly, ASAP 1 recovered 14 and ASAP 2 recovered 25, respectively.In C. adii sp.nov.C. assamensis, C. chinensis, and C. singhalanus, we found discrepancies between morphological and molecular delimitation analyses.Therefore, we compared the parameres of these species, which showed distinctness; however, species delimitation PTP, bPTP, and ASAP 1 indicated a single species, whereas ASAP 2 treated them as distinct species (Fig 4).
Similarly, we observed that C. esculentus sp.nov.forms a clade with C. chinensis and is closely related to it in terms of coloration.However, PTP and bPTP grouped this clade as a single species, while ASAP 1 and ASAP 2 treated C. esculentus sp.nov.and C. chinensis as separate species (Fig 4).Additionally, a closer look at the head and pygophore revealed that the major difference between C. esculentus sp.nov.and other species is in the shape of the mandibular plates and the parameres (Fig 12).It's important to note that the low node support for C. esculentus sp.nov.(PP: 0.82, MLBS: 52) is likely a result of the small sample size.A well-supported clade of C. nepalensis and two unidentified species resulted in a single species in the bPTP analysis.We could not examine the voucher specimens for unidentified species sequences, which create gaps in Fig 4. In the instance of C. ianus, all our analyses showed two putative species.C. ianus has several colour and size variants; it would be interesting to sequence multiple specimens to understand their relationships, but this remains out of the scope of the present study.The African species C. viduatus, which morphologically resembles C. ianus, formed a sister clade with each other (Fig 4).Our results also reveal that C. nigriventris, which was previously synonymized with C. nepalensis, forms a distinct lineage, and therefore we suggest treating that as a valid species.

Hidden diversity in Coridius chinensis
The current study shows that C. chinensis is a complex of species and, at the same time, disentangles some of its morphological diversity within north-eastern India.We found morphological variation along the geographical distribution of specimens of the C. chinensis complex and discovered at least six independent lineages, which were earlier grouped as one valid species, C. chinensis.The construction of trees using maximum likelihood and Bayesian inference for the several specimens of Coridius chinensis revealed similar tree topologies, with each clade being well-supported by high posterior probability and bootstrap support (see Fig 4).The phylogenetic placement of C. chinensis individuals may suggest that they are partially and geographically differentiated lineages of a species.
Our findings for C. chinensis indicate that there is high intraspecific diversity resulting in the six putative species.These individuals are from diverse geographic areas and may have different ecological niches and food preferences.Given this, we observed that C. chinensis is genetically diverse yet morphologically indistinguishable .Since the morphological investigation did not reveal any significant differences, we opted to maintain its current taxonomic status.To address the complexity within C. chinensis, future research will require larger taxon sampling across the occurrence regions, as well as multiple loci (nuclear and mitochondrial) data combined with ecological data.We agree that the use of additional nuclear gene markers can provide a well-resolved tree and gain a much deeper understanding of the phylogenetic relationships.

Taxonomic consideration
Durai [27] catalogued 37 species under the genus Coridius and listed all synonyms and distribution records for each species.Later, Lis [23] published a checklist of the Old World Dinidoridae species, including Coridius, along with a few new synonyms, and proposed two new genera: the first was Coridiellus Lis 1990, to which six species of Coridius were transferred, and the second was Colporidius Lis, where Coridius aeneus (Walker, 1868) was transferred.Considering all these changes, Rolston et.al. [42] published a catalogue of the world's Dinidoridae, which included only 32 valid species under the genus Coridius.
C. adii sp.nov.C. esculentus sp.nov.and C. insperatus sp.nov.described here, unquestionably belong to the genus Coridius since they exhibit all the key characters of Dinidorini and Coridius.Interestingly, according to Distant's [44] and Durai's [27] revisions, the genus Coridius has five segmented antennae.But C. insperatus sp.nov.described from Arunachal Pradesh, has only four segmented antennae that distinguish it from all other known species.The mandibular plates in C. insperatus sp.nov.are narrower and mesially meeting each other (Fig 9A), whereas in all other species they are broad and meet in front of the clypeus (Fig 9B -9K).
The Indian Coridius can be divided into two groups solely based on antennal coloration (though this has not been tested phylogenetically): the species with all antennal segments black form the first group, which includes C.  In 1997, Ahmad et. al. [48] described two new species of Coridius from Pakistan, C. turbatensis Ahmad, Hussain &Kamaluddin, 1997, andC. neobrunneus Ahmad, Hussain &Kamaluddin, 1997, with which we have compared the new species; however, these species are more closely related to C. ianus and C. brunneus (see [48]).
Distant [44] included 10 species under the genus Aspongopus (= Coridius), but later Durai [27] synonymized a few species, including C. nigriventris under C. nepalensis, based on the morphological similarity of aedeagus.In our analysis, we found that these two species formed distinct clades (Fig 4); hence, we reinstated and re-established the species status of C. nigriventris based on morphological and molecular evidence (Figs 7 and 8C) (for more information, refer to the results section).The most useful diagnostic characters for distinguishing species were revealed: these are relative size, length of antennal segments, shape of mandibular plates, pygophore, and parameres.
Finally, the discovery of C. adii sp.nov., C. esculentus sp.nov., and C. insperatus sp.nov.implies that there are likely many more species to be described from the north-eastern area of India.In addition, extensive systematic surveys throughout the range of C. chinensis are required to resolve the species' hidden diversity.

Conclusions
There are 13 Coridius species known from India, including three new species described in this study.The phylogenetic trees and species delimitation results support the establishment of three new Coridius species.The LDA revealed that the most important morphological characters to consider while identifying these species are total body length, maximum body width, and the length and width of the pronotum and scutellum.Our findings indicate that C. chinensis is a species complex that needs extensive research to resolve intraspecific variation.In this work, we reinstated C. nigriventris, which was previously synonymized under C. nepalensis.Also, we are providing illustrations of C. assamensis, C. fuscus, C. laosanus, C. sanguinolentus, and C. singhalanus for the first time.The current study on Coridius is intended to provide a framework for future studies with a larger sample size as well as to demonstrate that our integrative approach combining morphological (LDA) and molecular analyses (species delimitation methods) is most promising for resolving species delimitation and uncovering hidden diversity.The presence of several undescribed lineages in our data highlights the need to revise the taxonomy and systematics of Coridius.A large-scale global phylogenetic investigation of all known Coridius likely has great potential to yield numerous additional species.

Fig 1 .
Fig 1. Habitat and collection localities.(A) Natural habitat of Coridius spp. in Arunachal Pradesh.(B) Coridius spp. in a dry riverbed.(C) & (D) Collection localities of Coridius spp.across India.The map was created using QGIS Geographic Information System v3.28 by S. S. Boyane and does not include any copyrighted material.The SRTM file used in the map was obtained from the USGS server (public domain).https://doi.org/10.1371/journal.pone.0298176.g001

Fig 3 .
Fig 3. Linear Discriminant Analysis of six selected morphological characters based on the 10 species.The percentage separation achieved by the first two linear discriminant functions is 76.42% for axis 1 and 17.74% for axis 2, respectively.Because of the limited sample, C. adii sp.nov., and C. assamensis (Distant, 1902) were excluded from LDA. https://doi.org/10.1371/journal.pone.0298176.g003

Table
).Additionally, we retrieved 19 sequences of Coridius and 20 sequences belonging to Pentatomoidea and

Table 2 . Partition schemes and substitution models used for the Maximum likelihood (ML), Bayesian Inference (BI). Sl. no Gene RaxML Mr Bayes Length (bp)
MB), and the pronotum and scutellum's length and breadth ratios (PL/PB and SL/SB, respectively).The scatter diagram, histogram of the distribution of a single variable, and correlation values for each variable are provided in S1 and S2 Figs.