Species Delimitation Hypotheses Research Articles

An integrated approach to studying tropical plant diversity – Taxonomic monographs, herbarium specimens and the sweet potato

Recent decades have witnessed a massive increase in data accumulation, but our knowledge of the world’s biodiversity is still fragmentary: data accumulation has not been matched by a parallel taxonomic effort, and many groups of organisms have never been comprehensively studied. In the current context of climate change and biodiversity loss, we need to accelerate taxonomy and species discovery. This, however, requires a good taxonomic and phylogenetic framework, which is lacking for most groups of tropical plants. This contribution discusses the role of botanical monographs in accelerating taxonomy. We argue that the increasingly easier access to data in the world’s herbaria and the availability of DNA sequence data place botanists in an unparalleled position to produce taxonomic monographs, the forefront of taxonomic research. We illustrate the discussion with the results of a monographic study of the genus Ipomoea (Convolvulaceae). We integrated herbarium-based morphological studies with techniques of phylogenetic and genomic analysis of thousands of specimens to develop more robust species delimitation hypotheses and a comprehensive phylogenetic framework. Monographs such as ours have implications for other disciplines beyond taxonomy. We specifically show how it enabled important discoveries related to the origin of sweet potato, a worldwide staple crop.

Towards elucidating species diversity of European inland Strigamia (Chilopoda: Geophilomorpha): a first reassessment integrating multiple lines of evidence

Abstract Strigamia centipedes are widespread in European forest soils. However, a complex and inconsistent taxonomy has developed over time. Based on a modern species concept, we evaluated multiple lines of evidence for speciation among inland populations of Strigamia from the Italian region to the Baltic region, across central Europe. Hypotheses of species delimitation were drawn independently from: (1) morphological differentiation, by means of model-based cluster analyses of 52 specimens, after controlling for allometry and sexual dimorphism; (2) syntopy of morphologically distinct individuals, assumed to be representatives of coexistent species, from > 700 sites; and (3) molecular differentiation, by various methods applied to cytochrome c subunit I sequences of the same 52 specimens. Diagnoses and geographical distributions were revised by re-examining > 2000 specimens and the entire literature. We found preliminary evidence for: a Strigamia acuminata species complex, widespread, including a candidate species from the Eastern Alps differing in the forcipules; a Strigamia carniolensis species complex, hitherto often called Strigamia crassipes, widespread south of the Alps but absent from Sicily; a Strigamia crassipes species complex, hitherto often called Strigamia transsilvanica, more widespread than previously known, including three candidate species co-occurring in the Western Alps and differing in body size and the number of legs despite little genetic divergence; and Strigamia engadina, exclusive to a narrow part of the Central Alps.

A New Assessment of Robust Capuchin Monkey (Sapajus) Evolutionary History Using Genome-Wide SNP Marker Data and a Bayesian Approach to Species Delimitation.

Robust capuchin monkeys, Sapajus genus, are among the most phenotypically diverse and widespread groups of primates in South America, with one of the most confusing and often shifting taxonomies. We used a ddRADseq approach to generate genome-wide SNP markers for 171 individuals from all putative extant species of Sapajus to access their evolutionary history. Using maximum likelihood, multispecies coalescent phylogenetic inference, and a Bayes Factor method to test for alternative hypotheses of species delimitation, we inferred the phylogenetic history of the Sapajus radiation, evaluating the number of discrete species supported. Our results support the recognition of three species from the Atlantic Forest south of the São Francisco River, with these species being the first splits in the robust capuchin radiation. Our results were congruent in recovering the Pantanal and Amazonian Sapajus as structured into three monophyletic clades, though new morphological assessments are necessary, as the Amazonian clades do not agree with previous morphology-based taxonomic distributions. Phylogenetic reconstructions for Sapajus occurring in the Cerrado, Caatinga, and northeastern Atlantic Forest were less congruent with morphology-based phylogenetic reconstructions, as the bearded capuchin was recovered as a paraphyletic clade, with samples from the Caatinga biome being either a monophyletic clade or nested with the blond capuchin monkey.

Between a rock and a dry place: phylogenomics, biogeography, and systematics of ridge-tailed monitors (Squamata: Varanidae: Varanus acanthurus complex)

Genomic data are a powerful tool for the elucidation of evolutionary patterns at the population level and above. The combined analysis of genomic and morphological data can result in species delimitation hypotheses that reflect evolutionary history better than traditional taxonomy or any individual source of evidence. Here, we used thousands of single nucleotide polymorphisms, mitochondrial sequences, and comprehensive morphological data to characterize the evolutionary history of the ridge-tailed monitors in the Varanus acanthurus complex (V. acanthurus, V. baritji, and V. storri), a group of saxicolous lizards with a wide distribution in Australia, the driest vegetated continent. We found substantial genetic structure in the group and identify nine geographically clustered populations. Based on admixture patterns and species delimitation analyses we propose a taxonomic scheme that differs from current taxonomy. We consider V. acanthurus as monotypic, synonymize V. baritji with V. a. insulanicus (as a redefined V. insulanicus), elevate the subspecies of V. storri to full species (V. storri and V. ocreatus), and describe a new species from a previously identified center of endemism. The relationships among the species remain unresolved, likely as a result of fast speciation. Our study highlights the capability of large datasets to illuminate admixture patterns, biogeographic history, and species limits, even when phylogeny is not completely resolved. Furthermore, our results highlight the impact that the Cenozoic aridification of Australia had on saxicolous taxa and the role of mesic rocky escarpments as refugia. These habitats apparently allowed the persistence of lineages that became sources of colonization for arid environments.

Shedding light on species boundaries in small endogeic animals through an integrative approach: species delimitation in the centipedeClinopodes carinthiacus(Chilopoda: Geophilidae) in the south-eastern Alps

AbstractThe investigation of species boundaries in strictly endogeic animals is challenging because they are prone to fine-scale genetic and phenotypic geographical differentiation owing to low dispersal ability. An integrative approach exploiting different sources of information has seldom been adopted in these animals and even more rarely by treating all data sources equally. We investigated species boundaries in the endogeic centipede Clinopodes carinthiacus across the south-eastern Alps by studying genetic and morphological differentiation in a sample of 66 specimens from 27 sites, complemented by the morphological examination of more than 1100 specimens from other sites. Hypotheses of species delimitation were obtained independently from the molecular sequences of three markers (mitochondrial 16S and COI and nuclear 28S) by means of different species discovery methods (automatic barcode gap discovery, assemble species by automatic partitioning, general mixed Yule coalescent and the Poisson tree process) and from ten morphological characters by means of a model-based cluster analysis and Bayesian model selection. We found strong support for the existence of at least two species: C. carinthiacus s.s. and Clinopodes strasseri, which was formerly described as a subspecies of another species, and later placed in synonymy with C. carinthiacus. The two species coexist in syntopy in at least one site.

Delimitation despite discordance: Evaluating the species limits of a confounding species complex in the face of mitonuclear discordance.

The delimitation of species is an essential pursuit of biology, and proper taxonomies are crucial for the assessment and conservation management of organismal diversity. However, delimiting species can be hindered by a number of factors including highly conserved morphologies (e.g., cryptic species), differences in criteria of species concepts, lineages being in the early stages of the speciation or divergence process, and discordance between gene topologies (e.g., mitonuclear discordance). Here we use a taxonomically confounded species complex of toads in Central America that exhibits extensive mitonuclear discordance to test delimitation hypotheses. Our investigation integrates mitochondrial sequences, nuclear SNPs, morphology, and macroecological data to determine which taxonomy best explains the divergence and evolutionary relationships among these toads. We found that a three species taxonomy following the distributions of the nuclear SNP haplotypes offers the best explanation of the species in this complex based off of the integrated data types. Due to the taxonomic instability of this group, we also discuss conservation concerns in the face of improper taxonomic delimitation. Our study provides an empirical and integrative hypothesis testing framework to assess species delimitation hypotheses in the face of cryptic morphology and mitonuclear discordance and highlights the importance that a stable taxonomy has over conservation‐related actions.

Speciation by Sensory Drive in the Paragalago zanzibaricus Species Complex

Cryptic animal species are often distinguishable by nonvisual communication signals used in specific-mate recognition. The three cryptic taxa comprising the Paragalago zanzibaricus species complex use distinct loud calls that do not obviously reflect molecular phylogenetic relationships. Paragalago granti and P. cocos both have incremental calls but are not sister taxa; P. cocos is the sister taxon to P. zanzibaricus, which has a rolling (trilling) call like that of P. rondoensis, one of the outliers to the complex. To test current hypotheses of species delimitation, I conducted 378 playback trials, using conspecific and heterospecific loud calls and recording both vocal responses and movement toward the speaker. To investigate the sensory drive hypothesis (acoustic adaptation), I measured reverberation duration of 215 short “yap” mobbing calls and examined 282 sonotopes (local soundscapes). I tested the hypothesis that different species’ ear lengths allow the animals to filter out interfering frequencies in local background noise. On Zanzibar Island, P. zanzibaricus responded solely to conspecific calls (23/32). In South Africa, P. granti individuals responded to conspecific calls (12/22) but also sometimes to P. cocos calls (9/22). In Kenya, P. cocos individuals responded to conspecific calls (34/40) but also sometimes to P. zanzibaricus calls (8/40). The three habitats showed differences in reverberation duration, and soundscapes differed in background orthopteran frequencies. Incremental calls probably evolved in the ancestor of the complex, in response to high levels of reverberation and a continuous backdrop of katydid stridulation in dry forest. Secondary evolution of trilling and decreased ear size probably evolved as result of decreased reverberation and higher frequency katydid interference in the P. cocos–P. zanzibaricus ancestor. A loss of short units in P. zanzibaricus probably occurred as result of decreased reverberation in thicket. The study validates the three species of the complex and provides information supporting speciation by sensory drive.

Resolving the species status of overlooked West‐Palaearctic bumblebees

AbstractMultisource approaches in taxonomy gather different lines of evidence in order to draw strongly supported taxonomic conclusions and constitute the basis of integrative taxonomy. In the case of overlooked taxa with disjunct distributions for which sampling is more challenging, integrative approaches help to propose stable hypotheses at the species and subspecies levels. Here, based on genetic and semio‐chemical traits, we performed an integrative taxonomic analysis to evaluate species delimitation hypotheses within a monophyletic group of bumblebees (Hymenoptera, Apidae, Bombus) including the formerly recognised subgenera Eversmannibombus, Laesobombus and Mucidobombus which are now included in the subgenus Thoracobombus. Our results demonstrate the conspecificity of several polytypic taxa, and we formally recognise the subspecies Bombus laesus aliceae comb. nov. Cockerell, 1931, endemic to North Africa, based on its allopatry, unique mitochondrial haplotype and divergent cephalic labial gland secretions. This highlights the need to maintain studying polytypic complexes of bumblebee taxa for which phylogenetic relationships could be still entangled and eventually implement conservation strategies for taxonomically differentiated lineages.

Phylogenomic species delimitation in the ants of the Temnothorax salvini group (Hymenoptera: Formicidae): an integrative approach

ABSTRACTThe members of the Temnothorax salvini (Forel) species group are rarely collected, arboreally nesting ants of Central American forests. Previously thought to consist of two broadly dispersed species, recent collections have revealed a diversity of specimens that defy the two‐species salvini group concept, but these are difficult to distinguish from each other based solely on morphology. I contrast several model‐based approaches to species delimitation based on target‐enriched genomic data. With molecular data from thousands of ultraconserved elements (UCEs), mitochondrial genome sequences and morphometric data, I use an integrated approach to species delimitation within the salvini group. Morphometric data were analysed using cluster analysis of principal component analysis (PCA) output. I use several popular methods of molecular species delimitation, including bPTP, BPP and STACEY, using a novel approach to filtering UCE data based on posterior predictive checks of nucleotide substitution model adequacy. In addition, I use iBPP to integrate morphometric PCA data and filtered UCE data in a ‘total evidence’ analysis. I use geographical range data for an independent contrast to discriminate among competing species delimitation hypotheses. Furthermore, I investigate the evolutionary timescale and biogeographical history of the group and find that it arose roughly 13 Ma ago in habitats associated with present day mid‐to‐high elevations of the mountain complex spanning southern Mexico to northern Nicaragua. In addition, dispersal of the salvini group into the Southern Sierra Madre in Mexico, lowland habitats and the southern Central American cordilleras in Costa Rica and Panama subsequent to mountain building in southern Central America 5–8 Ma ago appears to follow a taxon‐cycle dynamic, with the lowland‐adapted T. aztecus representing the most recent expansion phase. I find that the salvini group, which previously contained two named species, is composed of nine, all of which are morphologically diagnosable a posteriori.

Another stripe on the tiger makes no difference? Unexpected diversity in the widespread tiger tarantula Davus pentaloris (Araneae: Theraphosidae: Theraphosinae)

Abstract Integrative taxonomy is relevant for the discovery and delimitation of cryptic species by incorporating different sources of evidence to construct rigorous species hypotheses. The genus Davus was recently revised and it was found that Davus pentaloris presents high morphological variation across its widespread distribution. However, tarantulas usually present low dispersal capabilities that occasionally result in local endemism. In order to evaluate species boundaries within this taxon, we examine the morphological variation and, additionally, employ four strategies based on mtDNA data (COI): two distance-based [automatic barcode gap discovery (ABGD) and Neighbor-Joining (NJ)] and two tree-based methods [general mixed Yule coalescent (GMYC) and Bayesian Poisson tree process (bPTP)]. Available morphological evidence recovers 13 putative morphospecies, but the different methods based on molecular data recover a variable number of candidate species (16–18). Based on the congruence across all analyses and the available morphological data, we recognize 13 clearly diagnosable species, 12 of them new to science. We conclude that the underestimation of the diversity in D. pentaloris was mainly caused by deficient practices in taxonomy, rather than by the presence of cryptic diversity. Although COI is a functional barcoding marker and it gives reliable results in our study, we recommend combining multiple sources of evidence and strategies to construct better species delimitation hypotheses.

Coalescence-based species delimitation using genome-wide data reveals hidden diversity in a cosmopolitan group of lichens

Although there is an abundance of species delimitation methods on the market, most approaches depend on predefined assignment of specimens to species or populations. Assignment-free methods, which can simultaneously infer boundaries and relationships among species, are of high importance in cases, when correct pre-assignment is difficult or not at all possible. In this study, we use assignment-free multispecies coalescent-based species delimitation (STACEY, tr2-delimitation, and BP&P), phylogenetic methods, and clustering algorithms to investigate the inter- and infraspecific relationships within a common and widespread group of lichens with contentious species boundaries. The Cetraria aculeata group presents a good example of extreme morphological variability and unclear species delimitation in lichens. Based on DNA-sequence data from 26 fungal loci and 10 microsatellite loci, as well as morphological and chemical data, our results provide evidence for the occurrence of five different taxa within the group and highlight the difficulties of morphologically distinguishing these species. We discovered a separate lineage (clade C) within C. aculeata s. str., which does not fully coincide with any of the a priori identified species C. aculeata, C. crespoae, or C. steppae and conclude that this clade constitutes a semi-cryptic, genetically isolated lineage within C. aculeata. We recognize this lineage at subspecific rank as C. aculeata subsp. steppae and synonymize Cetraria crespoae with C. aculeata subsp. aculeata. Epitypes are designated for all involved names to stabilize their usage. The PKS8 gene locus is recommended as a barcode for the separation of C. aculeata subsp. aculeata and subsp. steppae. We demonstrate the potential use of microsatellite data for species delimitation in lichens that might offer an alternative insight or be used to test species delimitation hypotheses, when dealing with closely related or potentially cryptic species. Our results also confirm the presence of an undescribed sister lineage to C. odontella previously misidentified as C. muricata and extend the known range of this lineage to Central Asia (Altay Mts.) and the Central European Alps (France, Switzerland), which calls for a critical reappraisal of records of C. aculeata and C. muricata from these mountain ranges.

Assessing the diversity of Western North American Juga (Semisulcospiridae, Gastropoda)

Juga is a genus of freshwater gastropods distributed in Pacific and Interior drainages of the Pacific Northwest from central California to northern Washington. The current classification has relied heavily on features of the shell, which vary within and across drainages, and often intergrade without sharp distinctions between species. The only previous molecular analysis included limited population sampling, which did not allow robust assessment of intra- versus interspecific levels of genetic diversity, and concluded almost every sampled population to be a distinct OTU. We assembled a multilocus mitochondrial (COI, 16S) and nuclear gene (ITS1) dataset for ∼100 populations collected across the range of the genus. We generated primary species hypotheses using ABGD with best-fit model-corrected distances and further explored our data, both individual gene partitions and concatenated datasets, using a diversity of phylogenetic and species delimitation methods (Bayesian inference, maximum likelihood estimation, StarBEAST2, bGMYC, bPTP, BP&P). Our secondary species delimitation hypotheses, based primarily on the criterion of reciprocal monophyly, and informed by a combination of geography and morphology, support the interpretation that Juga comprises a mixture of geographically widespread species and narrow range endemics. As might be expected in taxa with low vagility and poor dispersal capacities, analysis of molecular variance (AMOVA) revealed highly structured populations with up to 80% of the observed genetic variance explained by variation between populations. Analyses with bGMYC, bPTP, and BP&P appeared sensitive to this genetic structure and returned highly dissected species hypotheses that are likely oversplit. The species diversity of Juga is concluded to be lower than presently recognized, and the systematics to require extensive revision. Features of the teleoconch considered significant in species-level and subgeneric classification were found to be variable within some species, sometimes at a single site. Of a number of potentially new species identified in non-peer reviewed reports and field guides, only one was supported as a distinct OTU.

Integrating genomic and phenotypic data to evaluate alternative phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers.

Although resolving phylogenetic relationships and establishing species limits are primary goals of systematics, these tasks remain challenging at both conceptual and analytical levels. Here, we integrated genomic and phenotypic data and employed a comprehensive suite of coalescent-based analyses to develop and evaluate competing phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers (Chorthippus binotatus group) composed of two species and eight putative subspecies. To resolve the evolutionary relationships within this complex, we first evaluated alternative phylogenetic hypotheses arising from multiple schemes of genomic data processing and contrasted genetic-based inferences with different sources of phenotypic information. Second, we examined the importance of number of loci, demographic priors, number and kind of phenotypic characters and sex-based trait variation for developing alternative species delimitation hypotheses. The best-supported topology was largely compatible with phenotypic data and showed the presence of two clades corresponding to the nominative species groups, one including three well-resolved lineages and the other comprising a four-lineage polytomy and a well-differentiated sister taxon. Integrative species delimitation analyses indicated that the number of employed loci had little impact on the obtained inferences but revealed the higher power provided by an increasing number of phenotypic characters and the usefulness of assessing their phylogenetic information content and differences between sexes in among-taxa trait variation. Overall, our study highlights the importance of integrating multiple sources of information to test competing phylogenetic hypotheses and elucidate the evolutionary history of species complexes representing early stages of divergence where conflicting inferences are more prone to appear.

Integrative taxonomy of the neotropical genus Omalonyx (Elasmognatha: Succineidae)

The genus Omalonyx d'Orbigny, 1837, includes neotropical semi‐aquatic succineid slugs and comprises six recognized species to date. Field surveys across continental South America recovered five of the six recognized species. According to the morphological characters traditionally included in Omalonyx descriptions, the specimens were tentatively identified as O. matheroni, O. pattersonae, O. convexus, O. geayi and O. unguis. Employing sequences of mitochondrial cytochrome oxidase I (COI) alone or combined with the nuclear second internal transcribed spacer (ITS2) in discovery methods of species delimitation (GMYC and STACEY) led to species delimitation hypotheses that, except for unambiguously supporting O. convexus, have no correspondence to morphologically based assignments. To choose the delimitation model that best fit our data, the hypotheses recovered by GMYC, STACEY and morphology and created by merging species recovered by those methods had their marginal likelihood estimated and compared using the Bayes factors. The best‐supported hypothesis distinguished two species besides O. convexus: one widespread over most of South America and the other restricted to Rio de Janeiro State, Brazil. Furthermore, the pattern of genetic structuring supports pathways connecting the Amazonian and Atlantic forests. This pattern is similar to that observed in terrestrial taxa (e.g., forest‐dwelling small mammals) and is different from the pattern for fully aquatic taxa.

Following the cold: geographical differentiation between interglacial refugia and speciation in the arcto‐alpine species complex Bombus monticola (Hymenoptera: Apidae)

AbstractCold‐adapted species are expected to have reached their largest distribution range during a part of the Ice Ages whereas postglacial warming has led to their range contracting toward high‐latitude and high‐altitude areas. This has resulted in an extant allopatric distribution of populations and possibly to trait differentiations (selected or not) or even speciation. Assessing inter‐refugium differentiation or speciation remains challenging for such organisms because of sampling difficulties (several allopatric populations) and disagreements on species concept. In the present study, we assessed postglacial inter‐refugia differentiation and potential speciation among populations of one of the most common arcto‐alpine bumblebee species in European mountains, Bombus monticola Smith, 1849. Based on mitochondrial DNA/nuclear DNA markers and eco‐chemical traits, we performed integrative taxonomic analysis to evaluate alternative species delimitation hypotheses and to assess geographical differentiation between interglacial refugia and speciation in arcto‐alpine species. Our results show that trait differentiations occurred between most Southern European mountains (i.e. Alps, Balkan, Pyrenees, and Apennines) and Arctic regions. We suggest that the monticola complex actually includes three species: B. konradini stat.n. status distributed in Italy (Central Apennine mountains), B. monticola with five subspecies, including B. monticola mathildis ssp.n. distributed in the North Apennine mountains ; and B. lapponicus. Our results support the hypothesis that post‐Ice Age periods can lead to speciation in cold‐adapted species through distribution range contraction. We underline the importance of an integrative taxonomic approach for rigorous species delimitation, and for evolutionary study and conservation of taxonomically challenging taxa.

DNA barcoding and faunistic criteria for a revised taxonomy of Italian Ephemeroptera

Mayfly (Ephemeroptera) systematics has considerably changed over the years, but many questions have yet to be answered. The synergistic connection between traditional knowledge and new data sources, producing increasingly complex information, has become a compelling issue for modern taxonomy. Molecular tests and the use of reliable reference sequence libraries may constitute effective complements to the traditional method in guiding recognition of species and giving information about taxonomic incongruences which require further examination. In the present study, we sought to verify the current Italian mayfly nomenclatural system through DNA barcoding and relevant points to reliably manage the available amount of morpho-ecological and molecular data are discussed. We investigated COI (Cytochrome oxidase I) sequence variation in 163 individuals of Italian mayflies, 126 of which were previously assigned to 24 morphologically recognised species, and 37 could be attributed only to generic taxonomic entities (“sp.”, “cf.” or “gr.”). DNA barcoding statistical tests for species delimitation hypotheses based on genetic distances and inferred gene trees were integrated with GenBank searches and surveys of the historical literature to better understand the knowledge acquired on the status and diversity of the investigated taxa. Combined criteria to define three categories of reliability were then assessed. Concurrent data allowing unambiguous identification were attained for only eight species. High intraspecific genetic distances (> 3%) and a lack of reliable reference material or convincing taxonomic information evidenced 29 critical states, deserving further investigation. Solid species names, potential cryptic species and entities about which little is known are pointed out for a future upgrade/reorganisation of the taxonomy of Italian Ephemeroptera.

Validation and description of two new north-western Australian Rainbow skinks with multispecies coalescent methods and morphology.

While methods for genetic species delimitation have noticeably improved in the last decade, this remains a work in progress. Ideally, model based approaches should be applied and considered jointly with other lines of evidence, primarily morphology and geography, in an integrative taxonomy framework. Deep phylogeographic divergences have been reported for several species of Carlia skinks, but only for some eastern taxa have species boundaries been formally tested. The present study does this and revises the taxonomy for two species from northern Australia, Carlia johnstonei and C. triacantha. We introduce an approach that is based on the recently published method StarBEAST2, which uses multilocus data to explore the support for alternative species delimitation hypotheses using Bayes Factors (BFD). We apply this method, jointly with two other multispecies coalescent methods, using an extensive (from 2,163 exons) data set along with measures of 11 morphological characters. We use this integrated approach to evaluate two new candidate species previously revealed in phylogeographic analyses of rainbow skinks (genus Carlia) in Western Australia. The results based on BFD StarBEAST2, BFD* SNAPP and BPP genetic delimitation, together with morphology, support each of the four recently identified Carlia lineages as separate species. The BFD StarBEAST2 approach yielded results highly congruent with those from BFD* SNAPP and BPP. This supports use of the robust multilocus multispecies coalescent StarBEAST2 method for species delimitation, which does not require a priori resolved species or gene trees. Compared to the situation in C. triacantha, morphological divergence was greater between the two lineages within Kimberley endemic C. johnstonei, which also had deeper divergent histories. This congruence supports recognition of two species within C. johnstonei. Nevertheless, the combined evidence also supports recognition of two taxa within the more widespread C. triacantha. With this work, we describe two new species, Carlia insularis sp. nov and Carlia isostriacantha sp. nov. in the northwest of Australia. This contributes to increasing recognition that this region of tropical Australia has a rich and unique fauna.

Review of the Nassarius pauperus (Gould, 1850) complex (Nassariidae): Part 3, reinstatement of the genus Reticunassa, with the description of six new species

In this review (third part), several species within the Nassarius pauperus complex from the eastern Indian Ocean and western Pacific are treated, including a revised concept of Nassa paupera Gould, 1850, type species of the genus Reticunassa Iredale, 1936. In the most recent taxonomic revision, several species had been synonymized with Nassarius pauperus (Gould, 1850), despite distinctive differences among these species in shell morphology. We sequenced a fragment of the mitochondrial COI and the nuclear 28S genes of all available Nassarius pauperus complex species. Automatic barcode gap discovery and reciprocal monophyly were applied to propose species delimitation hypotheses and to support a new concept of the genus. Using morphological and molecular characters, Reticunassa is elevated to full genus rank. Six new species are described: Reticunassa visayaensis sp. nov., R. poppeorum sp. nov., R. annabolteae sp. nov., R. goliath sp. nov., R. intrudens sp. nov. and R. thailandensis sp. nov., while R. tringa (Souverbie, 1864) is recognized as a valid species.

Tephritid Integrative Taxonomy: Where We Are Now, with a Focus on the Resolution of Three Tropical Fruit Fly Species Complexes.

Accurate species delimitation underpins good taxonomy. Formalization of integrative taxonomy in the past decade has provided a framework for using multidisciplinary data to make species delimitation hypotheses more rigorous. We address the current state of integrative taxonomy by using as a case study an international project targeted at resolving three important tephritid species complexes: Bactrocera dorsalis complex, Anastrepha fraterculus complex, and Ceratitis FAR (C. fasciventris, C. anonae, C. rosa) complex. The integrative taxonomic approach has helped deliver significant advances in resolving these complexes: It has been used to identify some taxa as belonging to the same biological species as well as to confirm hidden cryptic diversity under a single taxonomic name. Nevertheless, the general application of integrative taxonomy has not been without issue, revealing challenges that must be considered when undertaking an integrative taxonomy project. Scrutiny of this international case study provides a unique opportunity to document lessons learned for the benefit of not only tephritid taxonomists, but also the wider taxonomic community.

RAD sequencing enables unprecedented phylogenetic resolution and objective species delimitation in recalcitrant divergent taxa

Species delimitations is problematic in many cases due to the difficulty of evaluating predictions from species hypotheses. In many cases delimitations rely on subjective interpretations of morphological and/or DNA data. Species with inadequate genetic resources needed to answer questions regarding evolutionary relatedness and genetic uniqueness are particularly problematic. In this study, we demonstrate the utility of restriction site associated DNA sequencing (RAD-seq) to objectively resolve unambiguous phylogenetic relationships in a recalcitrant group of deep-sea corals with divergences >80 million years. We infer robust species boundaries in the genus Paragorgia by testing alternative delimitation hypotheses using a Bayes Factors delimitation method. We present substantial evidence rejecting the current morphological species delimitation model for the genus and infer the presence of cryptic species associated with environmental variables. We argue that the suitability limits of RAD-seq for phylogenetic inferences cannot be assessed in terms of absolute time, but are contingent on taxon-specific factors. We show that classical taxonomy can greatly benefit from integrative approaches that provide objective tests to species delimitation hypotheses. Our results lead the way for addressing further questions in marine biogeography, community ecology, population dynamics, conservation, and evolution.