Finding complexity in complexes: Assessing the causes of mitonuclear discordance in a problematic species complex of Mesoamerican toads

Mitonuclear discordance is a frequently encountered pattern in phylogeographic studies and occurs when mitochondrial and nuclear DNA display conflicting signals. Discordance among these genetic markers can be caused by several factors including confounded taxonomies, gene flow, and incomplete lineage sorting. In this study, we present a strong case of mitonuclear discordance in a species complex of toads (Bufonidae: Incilius coccifer complex) found in the Chortís Block of Central America. To determine the cause of mitonuclear discordance in this complex, we used spatially explicit genetic data to test species limits and relationships, characterize demographic history, and quantify gene flow. We found extensive mitonuclear discordance among the three recognized species within this group, especially in populations within the Chortís Highlands of Honduras. Our data reveal nuclear introgression within the Chortís Highlands populations that was most probably driven by cyclical range expansions due to climatic fluctuations. Though we determined introgression occurred within the nuclear genome, our data suggest that it is not the key factor in driving mitonuclear discordance in the entire species complex. Rather, due to a lack of discernible geographic pattern between mitochondrial and nuclear DNA, as well as a relatively recent divergence time of this complex, we concluded that mitonuclear discordance has been caused by incomplete lineage sorting. Our study provides a framework to test sources of mitonuclear discordance and highlights the importance of using multiple marker types to test species boundaries in cryptic species.     

Mitonuclear discordance in wolf spiders: Genomic evidence for species integrity and introgression

Systematists and taxonomists have benefited greatly from the emergence of molecular methods. Species identification has become straightforward through DNA barcoding and the rapid build‐up of massive DNA barcode reference libraries. In animals, mitonuclear discordance can significantly complicate the process of species identification and delimitation. The causes of mitonuclear discordance are either biological (e.g., introgression, incomplete lineage sorting, horizontal gene transfer androgenesis) or induced by operational factors (e.g., human error with specimen misidentification or incorrect species delimitation). Moreover, endosymbionts may play an important role in promoting fixation of mitochondrial genomes. Here, we study the mitonuclear discordance of wolf spiders species (Lycosidae) (independent cases from Alopecosa aculeata and Pardosa pullata groups) that share identical COI DNA barcodes. We approached the case utilizing double‐digest restriction site‐associated DNA sequencing (ddRADseq) to obtain and analyse genomic‐scale data. Our results suggest that the observed cases of mitonuclear discordance are not due to operational reasons but result from biological processes. Further analysis indicated introgression and that incomplete lineage sorting is unlikely to have been responsible for the observed discrepancy. Additional survey of endosymbionts provided ideas on further research and their role in shaping mitochondrial DNA distribution patterns. Thus, ddRADseq grants an efficient way to study the taxonomy of problematic groups with insight into underlying evolutionary processes.     

Mitonuclear discordance as a confounding factor in the DNA taxonomy of monogonont rotifers

Discordance between mitochondrial and nuclear phylogenies is being increasingly recognized in animals and may confound DNA‐based taxonomy. This is especially relevant for taxa whose microscopic size often challenges any effort to distinguish between cryptic species without the assistance of molecular data. Regarding mitonuclear discordance, two strikingly contrasting scenarios have been recently demonstrated in the monogonont rotifers of the genus Brachionus. While strict mitonuclear concordance was observed in the marine B. plicatilis species complex, widespread hybridization‐driven mitonuclear discordance was revealed in the freshwater B. calyciflorus species complex. Here, we investigated the frequency of occurrence and the potential drivers of mitonuclear discordance in three additional freshwater monogonont rotifer taxa, and assessed its potential impact on the reliability of DNA taxonomy results based on commonly used single markers. We studied the cryptic species complexes of Keratella cochlearis, Polyarthra dolichoptera and Synchaeta pectinata. Phylogenetic reconstructions were based on the mitochondrial barcoding marker cytochrome c oxidase subunit I gene and the nuclear internal transcribed spacer 1 locus, which currently represent the two most typical genetic markers used in rotifer DNA taxonomy. Species were delimited according to each marker separately using a combination of tree‐based coalescent, distance‐based and allele‐sharing‐based approaches. Mitonuclear discordance was observed in all species complexes with incomplete lineage sorting and unresolved phylogenetic reconstructions recognized as the likely drivers. Evidence from additional sources, such as morphology and ecology, is thus advisable for deciding between often contrasting mitochondrial and nuclear species scenarios in these organisms.     

Species limits in butterflies (Lepidoptera: Nymphalidae): reconciling classical taxonomy with the multispecies coalescent

Species delimitation is at the core of biological sciences. During the last decade, molecular‐based approaches have advanced the field by providing additional sources of evidence to classical, morphology‐based taxonomy. However, taxonomy has not yet fully embraced molecular species delimitation beyond threshold‐based, single‐gene approaches, and taxonomic knowledge is not commonly integrated into multilocus species delimitation models. Here we aim to bridge empirical data (taxonomic and genetic) with recently developed coalescent‐based species delimitation approaches. We use the multispecies coalescent model as implemented in two Bayesian methods (dissect/stacey and bp&p ) to infer species hypotheses. In both cases, we account for phylogenetic uncertainty (by not using any guide tree) and taxonomic uncertainty (by measuring the impact of using a priori taxonomic assignments to specimens). We focus on an entire Neotropical tribe of butterflies, the Haeterini (Nymphalidae: Satyrinae). We contrast divergent taxonomic opinion – splitting, lumping and misclassifying species – in the light of different phenotypic classifications proposed to date. Our results provide a solid background for the recognition of 22 species. The synergistic approach presented here overcomes limitations in both traditional taxonomy (e.g. by recognizing cryptic species) and molecular‐based methods (e.g. by recognizing structured populations, and not raising them to species). Our framework provides a step forward towards standardization and increasing reproducibility of species delimitations.     

The Role of Integrative Taxonomy in the Conservation Management of Cryptic Species: The Taxonomic Status of Endangered Earless Dragons (Agamidae: Tympanocryptis) in the Grasslands of Queensland,Australia

Molecular phylogenetics is increasingly highlighting the prevalence of cryptic species, where morphologically similar organisms have long independent evolutionary histories. When such cryptic species are known to be declining in numbers and are at risk of extinction due to a range of threatening processes, the disjunction between molecular systematics research and conservation policy becomes a significant problem. We investigate the taxonomic status of Tympanocryptis populations in Queensland, which have previously been assigned to T. tetraporophora, using three species delimitation approaches. The taxonomic uncertainties in this species-group are of particular importance in the Darling Downs Earless Dragon (T. cf. tetraporophora), which is ranked as an endangered ‘species’ of high priority for conservation by the Queensland Department of Environment and Heritage Protection. We undertook a morphological study, integrated with a comprehensive genetic study and species delimitation analyses, to investigate the species status of populations in the region. Phylogenetic analyses of two gene regions (mtDNA: ND2; nuclear: RAG1) revealed high levels of genetic divergence between populations, indicating isolation over long evolutionary time frames, and strongly supporting two independent evolutionary lineages in southeastern Queensland, from the Darling Downs, and a third in the Gulf Region of northern Queensland. Of the three species delimitation protocols used, we found integrative taxonomy the most applicable to this cryptic species complex. Our study demonstrates the utility of integrative taxonomy as a species delimitation approach in cryptic complexes of species with conservation significance, where limited numbers of specimens are available.     

A mirage of cryptic species: Genomics uncover striking mitonuclear discordance in the butterfly Thymelicus sylvestris

Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double‐digest restriction‐site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex‐biased asymmetries, NUMTs, natural selection, introgression or Wolbachia‐mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events.     

Multilocus phylogeny and Bayesian estimates of species boundaries reveal hidden evolutionary relationships and cryptic diversity in Southeast Asian monitor lizards

Recent conceptual, technological and methodological advances in phylogenetics have enabled increasingly robust statistical species delimitation in studies of biodiversity. As the variety of evidence purporting species diversity has increased, so too have the kinds of tools and inferential power of methods for delimiting species. Here, we showcase an organismal system for a data‐rich, comparative molecular approach to evaluating strategies of species delimitation among monitor lizards of the genus Varanus. The water monitors (Varanus salvator Complex), a widespread group distributed throughout Southeast Asia and southern India, have been the subject of numerous taxonomic treatments, which have drawn recent attention due to the possibility of undocumented species diversity. To date, studies of this group have relied on purportedly diagnostic morphological characters, with no attention given to the genetic underpinnings of species diversity. Using a 5‐gene data set, we estimated phylogeny and used multilocus genetic networks, analysis of population structure and a Bayesian coalescent approach to infer species boundaries. Our results contradict previous systematic hypotheses, reveal surprising relationships between island and mainland lineages and uncover novel, cryptic evolutionary lineages (i.e. new putative species). Our study contributes to a growing body of literature suggesting that, used in concert with other sources of data (e.g. morphology, ecology, biogeography), multilocus genetic data can be highly informative to systematists and biodiversity specialists when attempting to estimate species diversity and identify conservation priorities. We recommend holding in abeyance taxonomic decisions until multiple, converging lines of evidence are available to best inform taxonomists, evolutionary biologists and conservationists.     

Genomic data reveal two distinct species from the widespread alpine ginger Roscoea tibetica Batalin (Zingiberaceae)

Species delimitation is a key foundation for exploring biodiversity. However, the existence of continuous phenotypic variation in widespread species challenges accurate species delimitation based on classical taxonomy. In this study, we investigated the cryptic diversity of a widespread herb (Roscoea tibetica Batalin) in a biodiversity hotspot (the Hengduan Mountains, China) using genotyping by sequencing, examining morphological traits, developing species distribution models, and simulating demographic history. Phylogenomic reconstruction, principal component analysis, and genetic structure inferences indicated that previously reported R. tibetica comprised two monophyletic lineages with a deep divergence. Several morphological diagnostic characteristics were discovered from field and common garden that corresponded to these independent evolutionary lineages. Species distribution models illustrated significant ecological divergence between both lineages. All evidence strongly supported that R. tibetica, as described in previous taxonomy, actually comprises two distinct species. Model test of gene flow and effective population size changes in fastsimcoal2, and a negative Tajima's D-value suggested that recent contact likely occurred between the two lineages. Our results proposed that cryptic diversity in previously reported R. tibetica was possibly associated with phenotypic plasticity in heterogeneous environments and morphological convergence in similar habitats. This study suggests that caution should be exercised when attempting to gain biological insight into species with large-scale morphological variation, and species delimitation should be done in advance.     

The toad’s warts: Discordance creates bumpy expectations of mitochondrial‐nuclear evolution between species

Discordance between the mitochondrial and nuclear genomes is a prevalent phenomenon in nature, in which the underlying processes responsible are considered to be important in shaping genetic variation in natural populations. Among the evolutionary processes that best explain such genomic mismatches incomplete lineage sorting and introgression are commonly identified, however, many studies are unable to distinguish between these hypotheses, which has become a major challenge in the field. In this issue of Molecular Ecology, Firneno et al. (2020) present an elegant exploration of mitochondrial‐nuclear discordance in Mesoamerican toads. Integrating genome‐scale and spatial data to test between these hypotheses within an empirical model testing framework, they find strong support that incomplete lineage sorting explains the observed discordance. Their work, along with many previous articles in Molecular Ecology, highlights the commonality of mito‐nuclear discordance among species despite the expectations of tightly concerted mitochondrial and nuclear genome evolution. It is increasingly clear that the nuclear genomes of many species are (at least for short periods of evolutionary time) functionally compatible with multiple, divergent mitochondrial haplotypes. As such, we suggest future research not only seeks to understand the processes causing spatial mito‐nuclear discordance (e.g. incomplete lineage sorting, introgression), but also explores those that maintain discordance through time and space (e.g. relaxed selection on mito‐nuclear interactions, heterozygosity, population demographics). We also discuss the vital role that taxonomy plays in interpreting patterns of mito‐nuclear discordance when data‐consistent yet differing taxonomies are used, such as treating allopatrically distributed taxa as multiple isolated populations versus multiple micro‐endemic species.     

Species delimitation of the white‐tailed rubythroat Calliope pectoralis complex (Aves,Muscicapidae) using an integrative taxonomic approach

Our knowledge of the systematics and taxonomy of Asian birds has improved much in the last two decades, and the number of recognised species has increased significantly as a result of in‐depth studies using an integrative taxonomic approach. The Sino‐Himalayan mountains harbor a high level of passerine diversity. Several allopatric or parapatric taxa that are currently treated as subspecies of polytypic species within that region are likely to deserve full species status, and thus their taxonomic status needs to be revisited. Based on analyses of multilocus data, vocalizations and morphology, we propose that the white‐tailed rubythroat Calliope pectoralis should be treated as two species, the Himalayan rubythroat C. pectoralis sensu stricto in the Tian Shan and Himalayan mountains, and the Chinese rubythroat C. tschebaiewi in the mountains of southwestern and north‐central China. According to our dating analyses based on mitochondrial loci, these two species diverged approximately 2.2 million yr ago. We further found that C. tschebaiewi was paraphyletic to C. pectoralis sensu stricto in nuclear data, which demonstrates a state of mitonuclear discordance that warrants further work. Our results suggest that geographic changes and glacial cycles in the Pleistocene may have caused allopatric divergence in the C. pectoralis complex. Our study stresses the importance of applying an integrative taxonomy approach to fully unravel the true avian diversity in the Sino‐Himalayan Mountains.     

Rigorous approaches to species delimitation have significant implications for African crocodilian systematics and conservation

Accurate species delimitation is a central assumption of biology that, in groups such as the Crocodylia, is often hindered by highly conserved morphology and frequent introgression. In Africa, crocodilian systematics has been hampered by complex regional biogeography and confounded taxonomic history. We used rigorous molecular and morphological species delimitation methods to test the hypothesis that the slender-snouted crocodile (Mecistops cataphractus) is composed of multiple species corresponding to the Congolian and Guinean biogeographic zones. Speciation probability was assessed by using 11 mitochondrial and nuclear genes, and cranial morphology for over 100 specimens, representing the full geographical extent of the species distribution. Molecular Bayesian and phylogenetic species delimitation showed unanimous support for two Mecistops species isolated to the Upper Guinean and Congo (including Lower Guinean) biomes that were supported by 13 cranial characters capable of unambiguously diagnosing each species. Fossil-calibrated phylogenetic reconstruction estimated that the species split ± 6.5–7.5 Ma, which is congruent with intraspecies divergence within the sympatric crocodile genus Osteolaemus and the formation of the Cameroon Volcanic Line. Our results underscore the necessity of comprehensive phylogeographic analyses within currently recognized taxa to detect cryptic species within the Crocodylia. We recommend that the community of crocodilian researchers reconsider the conceptualization of crocodilian species especially in the light of the conservation ramifications for this economically and ecologically important group.     

Integrative taxonomy and conservation of cryptic beetles in the Mediterranean region (Hydrophilidae)

Arribas, P., Andújar, C., Sánchez‐Fernández, D., Abellán, P. & Millán, A. (2012). Integrative taxonomy and conservation of cryptic beetles in the Mediterranean region (Hydrophilidae). —Zoologica Scripta, 00, 000–000. Because biodiversity inventory forms the basis for the effective conservation of species and habitats, there is an imperative need to discover and describe new species. A significant part of presently unknown biodiversity is constituted by cryptic species complexes, where traditional taxonomy usually fails due to a lack of clear taxonomic characters in the external structures of specimens. Integrative taxonomy offers a powerful tool to shed light on this part of encrypted biodiversity, combining multiple operational criteria in an evolutionary context in order to delineate species boundaries. The present study used an integrative approach to explore the species boundaries in a water beetle complex (Enochrus falcarius species complex) inhabiting saline streams, a rare and threatened habitat across the Mediterranean region. First, hypotheses about the candidate species on the basis of phylogenetic analyses and biogeographical information were proposed. Second, lineage divergence was evaluated between candidate species using (i) molecular cluster delimitation, (ii) morphometry (both linear body morphometrics and pronotum outlines) and (iii) ecological niche similarity estimates. We found divergence between candidate species on the basis of molecular, biogeographical and niche information, and consequently, four species were delimited within the E. falcarius complex (i.e. Enochrus jesusarribasi sp. n., Enochrus blazquezae sp. n., Enochrus risii sp. n. and Enochrus falcarius), despite the fact that they showed high morphological similarity. Enochrus falcarius, as previously considered, had not been proposed to be of conservational concern, because until now, it had been regarded as a single broadly distributed species in the Mediterranean region. However, the four entities here delimited within this species complex displayed characteristics that categorised them as vulnerable taxa. Hence, these results show how applying integrative taxonomy approaches and rapid vulnerability assessments to lineages from threatened habitats with the potential to comprise cryptic diversity could become a fundamental tool for biodiversity conservation, driving the discovery of cryptic species and consequently the modification of previous, inadequately evaluated vulnerability categorisations.     

An evaluation of candidate plant DNA barcodes and assignment methods in diagnosing 29 species in the genus Agalinis (Orobanchaceae)

? Premise of the study: DNA barcoding has been proposed as a useful technique within many disciplines (e.g., conservation biology and forensics) for determining the taxonomic identity of a sample based on nucleotide similarity to samples of known taxonomy. Application of DNA barcoding to plants has primarily focused on evaluating the success of candidate barcodes across a broad spectrum of evolutionary divergence. Less attention has been paid to evaluating performance when distinguishing congeners or to differential success of analytical techniques despite the fact that the practical application and utility of barcoding hinges on the ability to distinguish closely related species. ? Methods: We tested the ability to distinguish among 92 samples representing 29 putative species in the genus Agalinis (Orobanchaceae) using 13 candidate barcodes and three analytical methods (i.e., threshold genetic distances, hierarchical tree-based, and diagnostic character differences). Due to questions regarding evolutionary distinctiveness of some taxa, we evaluated success under two taxonomic hypotheses. ? Key results: The psbA-trnH and trnT-trnL barcodes in conjunction with the "best close match" distance-based method best met the objectives of DNA barcoding. Success was also a function of the taxonomy used. ? Conclusions: In addition to accurately identifying query sequences, our results showed that DNA barcoding is useful for detecting taxonomic uncertainty; determining whether erroneous taxonomy or incomplete lineage sorting is the cause requires additional information provided by traditional taxonomic approaches. The magnitude of differentiation within and among the Agalinis species sampled suggests that our results inform how DNA barcoding will perform among closely related species in other genera.     

Quantitative criteria for species delimitation

Species are the fundamental units of biology, ecology and conservation, and progress in these fields is therefore hampered by widespread taxonomic bias and uncertainty. Numerous operational techniques based on molecular or phenotypic data have been designed to overcome this problem, yet existing procedures remain subjective or inconsistent, particularly when applying the biological species concept. We address this issue by developing quantitative methods for a classic technique in systematic zoology, namely the use of divergence between undisputed sympatric species as a yardstick for assessing the taxonomic status of allopatric forms. We calculated mean levels of differentiation in multiple phenotypic characters – including biometrics, plumage and voice – for 58 sympatric or parapatric species‐pairs from 29 avian families. We then used estimates of mean divergence to develop criteria for species delimitation based on data‐driven thresholds. Preliminary tests show that these criteria result in relatively few changes to avian taxonomy in Europe, yet are capable of extensive reassignment of species limits in poorly known tropical regions. While we recognize that species limits are in many cases inherently arbitrary, we argue that our system can be applied to the global avifauna to deliver taxonomic decisions with a high level of objectivity, consistency and transparency.     

Detecting cryptic species in sympatry and allopatry: analysis of hidden diversity in Polyommatus (Agrodiaetus) butterflies (Lepidoptera: Lycaenidae)

Modern multilocus molecular techniques are a powerful tool in the detection and analysis of cryptic taxa. However, its shortcoming is that with allopatric populations it reveals phylogenetic lineages, not biological species. The increasing power of coalescent multilocus analysis leads to the situation in which nearly every geographically isolated or semi‐isolated population can be identified as a lineage and therefore raised to species rank. It leads to artificial taxonomic inflation and as a consequence creates an unnecessary burden on the conservation of biodiversity. To solve this problem, we suggest combining modern lineage delimitation techniques with the biological species concept. We discuss several explicit principles on how genetic markers can be used to detect cryptic entities that have properties of biological species (i.e. of actually or potentially reproductively isolated taxa). Using these principles we rearranged the taxonomy of the butterfly species close to Polyommatus (Agrodiaetus) ripartii. The subgenus Agrodiaetus is a model system in evolutionary research, but its taxonomy is poorly elaborated because, as a rule, most of its species are morphologically poorly differentiated. The taxon P. (A.) valiabadi has been supposed to be one of the few exceptions from this rule due to its accurately distinguishable wing pattern. We discovered that in fact traditionally recognized P. valiabadi is a triplet of cryptic species, strongly differentiated by their karyotypes and mitochondrial haplotypes.     

(New) Species concepts,species delimitation and the inherent limitations of taxonomy

The species problem, despite decades of heated debates, has not been resolved yet. Recently, two new species concepts have been published, the mitonuclear compatibility species concept and the inclusive species concept. I briefly discuss them, together with a recent attempt at standardizing taxonomic decisions, in the broader framework of what I believe is an inherent limitation of taxonomy—imposing a discrete system on a continuous process (evolution) that leads to fuzzy boundaries in nature. In the light of this, taxonomists, biologists in general and conservationists alike will have to accept the fact that completely nonarbitrary species delimitation is impossible. This has serious ramifications in all disciplines that rely on species, and particularly species counts, as a basic currency for quantitative analyses (ecology, evolutionary biology) and practical decision-making (conservation and environmental policy).     

Species boundaries in the messy middle—A genome‐scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi

Species delimitation among closely related species is challenging because traditional phenotype‐based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high‐throughput sequencing, it has become increasingly cost‐effective to acquire genome‐scale data which can resolve previously ambiguous species boundaries. As the availability of genome‐scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro‐endemism in a lineage of lichen‐forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single‐locus and genome‐scale data consisting of (a) single‐locus species delimitation analysis using ASAP, (b) maximum likelihood‐based phylogenetic tree inference, (c) genome‐scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross‐validate results between genome‐scale and single‐locus datasets, differently sampled subsets of genomic data and to control for population‐level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro‐endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.     

Coalescent Method in Conjunction with Niche Modeling Reveals Cryptic Diversity among Centipedes in the Western Ghats of South India

Background

There has been growing interest in integrative taxonomy that uses data from multiple disciplines for species delimitation. Typically, in such studies, monophyly is taken as a proxy for taxonomic distinctiveness and these units are treated as potential species. However, monophyly could arise due to stochastic processes. Thus here, we have employed a recently developed tool based on coalescent approach to ascertain the taxonomic distinctiveness of various monophyletic units. Subsequently, the species status of these taxonomic units was further tested using corroborative evidence from morphology and ecology. This inter-disciplinary approach was implemented on endemic centipedes of the genus Digitipes (Attems 1930) from the Western Ghats (WG) biodiversity hotspot of India. The species of the genus Digitipes are morphologically conserved, despite their ancient late Cretaceous origin.

Principal Findings

Our coalescent analysis based on mitochondrial dataset indicated the presence of nine putative species. The integrative approach, which includes nuclear, morphology, and climate datasets supported distinctiveness of eight putative species, of which three represent described species and five were new species. Among the five new species, three were morphologically cryptic species, emphasizing the effectiveness of this approach in discovering cryptic diversity in less explored areas of the tropics like the WG. In addition, species pairs showed variable divergence along the molecular, morphological and climate axes.

Conclusions

A multidisciplinary approach illustrated here is successful in discovering cryptic diversity with an indication that the current estimates of invertebrate species richness for the WG might have been underestimated. Additionally, the importance of measuring multiple secondary properties of species while defining species boundaries was highlighted given variable divergence of each species pair across the disciplines.