A Good Compromise: Rapid and Robust Species Proxies for Inventorying Biodiversity Hotspots Using the Terebridae (Gastropoda: Conoidea)

Devising a reproducible approach for species delimitation of hyperdiverse groups is an ongoing challenge in evolutionary biology. Speciation processes combine modes of passive and adaptive trait divergence requiring an integrative taxonomy approach to accurately generate robust species hypotheses. However, in light of the rapid decline of diversity on Earth, complete integrative approaches may not be practical in certain species-rich environments. As an alternative, we applied a two-step strategy combining ABGD (Automated Barcode Gap Discovery) and Klee diagrams, to balance speed and accuracy in producing primary species hypotheses (PSHs). Specifically, an ABGD/Klee approach was used for species delimitation in the Terebridae, a neurotoxin-producing marine snail family included in the Conoidea. Delimitation of species boundaries is problematic in the Conoidea, as traditional taxonomic approaches are hampered by the high levels of variation, convergence and morphological plasticity of shell characters. We used ABGD to analyze gaps in the distribution of pairwise distances of 454 COI sequences attributed to 87 morphospecies and obtained 98 to 125 Primary Species Hypotheses (PSHs). The PSH partitions were subsequently visualized as a Klee diagram color map, allowing easy detection of the incongruences that were further evaluated individually with two other species delimitation models, General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). GMYC and PTP results confirmed the presence of 17 putative cryptic terebrid species in our dataset. The consensus of GMYC, PTP, and ABGD/Klee findings suggest the combination of ABGD and Klee diagrams is an effective approach for rapidly proposing primary species proxies in hyperdiverse groups and a reliable first step for macroscopic biodiversity assessment.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.     

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with morphology

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species‐specific in spiders – exhibits considerable intraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu & Kuntner, 2015, endemic to south‐east China, where males are only available for two of the seven morphological species (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI) for 51 newly collected specimens of six morphological species and analysed them using five species‐delimitation methods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD), generalized mixed Yule‐coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, completes the revision of the genus. Although multi‐locus phylogenetic approaches may be needed for complex taxonomic delimitations, our results indicate that even single‐locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation. © 2015 The Linnean Society of London     

Species detection and individual assignment in species delimitation: can integrative data increase efficacy?

Statistical species delimitation usually relies on singular data, primarily genetic, for detecting putative species and individual assignment to putative species. Given the variety of speciation mechanisms, singular data may not adequately represent the genetic, morphological and ecological diversity relevant to species delimitation. We describe a methodological framework combining multivariate and clustering techniques that uses genetic, morphological and ecological data to detect and assign individuals to putative species. Our approach recovers a similar number of species recognized using traditional, qualitative taxonomic approaches that are not detected when using purely genetic methods. Furthermore, our approach detects groupings that traditional, qualitative taxonomic approaches do not. This empirical test suggests that our approach to detecting and assigning individuals to putative species could be useful in species delimitation despite varying levels of differentiation across genetic, phenotypic and ecological axes. This work highlights a critical, and often overlooked, aspect of the process of statistical species delimitation—species detection and individual assignment. Irrespective of the species delimitation approach used, all downstream processing relies on how individuals are initially assigned, and the practices and statistical issues surrounding individual assignment warrant careful consideration.     

Population structure and species boundary delimitation of cryptic Dioryctria moths: an integrative approach

Accurate delimitation of species boundaries is especially important in cryptic taxa where one or more character sources are uninformative or are in conflict. Rather than relying on a single marker to delimit species, integrative taxonomy uses multiple lines of evidence such as molecular, morphological, behavioural and geographic characters to test species limits. We examine the effectiveness of this approach by testing the delimitation of two cryptic Nearctic species of Dioryctria (Lepidoptera: Pyralidae) using three independent molecular markers [cytochrome c oxidase I (COI), second internal transcribed spacer unit (ITS2), and elongation factor 1alpha (EF1alpha)], forewing variation and larval host plant association. Although mitochondrial DNA (mtDNA) haplotypes do not form reciprocally monophyletic clades, restricted gene flow between COI haplotype groups, and concordance with ITS2 genotypes, forewing variation and host plant associations support delimitation of two Nearctic species: eastern Dioryctria reniculelloides and western Dioryctria pseudotsugella. Conversely, EF1alpha genotype variation was incongruent with the two previous markers. A case of discordance between COI and ITS2 was detected, suggesting either introgression due to hybridization or retained ancestral polymorphism due to incomplete coalescence. This study is consistent with other similar literature where molecular loci in closely related species progress from shared to fixed haplotypes/alleles, and from polyphyletic to reciprocally monophyletic relationships, although loci may vary in these characteristics despite maintenance of genomic integrity between distinct species. In particular, mtDNA in other studies generally showed a lower rate of fixation of differences than did X-linked or autosomal loci, reinforcing the need to use an integrative approach for delimiting species.     

Quantitative morphology and species delimitation under the general lineage concept: Optimization for Hedera (Araliaceae)

? Premise of the study: The use of continuous morphological characters in taxonomy is traditionally contingent on the existence of discrete diagnostic characters. When plant species are the result of recent divergence and gene flow and/or hybridization occur, the use of continuous morphological characters may help in species identification and delimitation. Between nine and 15 species have been recognized in the last treatments of Hedera. The recent divergence of the species and the involvement of allopolyploidization as the main force in this process may have greatly impeded the establishment of clear limits and contributed to multiple taxonomic proposals. ? Methods: A multivariate statistical decision-making procedure was applied to 56 quantitative morphological characters and 602 specimens to identify and delimit Hedera species under the general lineage concept. Species' exclusive genetic ancestry was evaluated with the genealogical sorting index from the Bayesian inference trees of 30 Hedera ITS sequences. ? Key results: The decision-making procedure allowed recognizing 12 species and two groups (stellate and scale-like trichome groups) in Hedera and provided statistical support for making decisions about long-standing taxonomic controversies. Common ancestry was detected for the populations of three species even in the absence of the species monophyly. ? Conclusions: Quantitative variation supports discrete variation and provides statistical support for the taxa recognized in some recent proposals of Hedera. The need of explicit analysis of quantitative data are claimed to reduce taxonomic subjectivity and ease decision-making when qualitative data fail.     

A Retrospective Approach to Testing the DNA Barcoding Method

A decade ago, DNA barcoding was proposed as a standardised method for identifying existing species and speeding the discovery of new species. Yet, despite its numerous successes across a range of taxa, its frequent failures have brought into question its accuracy as a short-cut taxonomic method. We use a retrospective approach, applying the method to the classification of New Zealand skinks as it stood in 1977 (primarily based upon morphological characters), and compare it to the current taxonomy reached using both morphological and molecular approaches. For the 1977 dataset, DNA barcoding had moderate-high success in identifying specimens (78-98%), and correctly flagging specimens that have since been confirmed as distinct taxa (77-100%). But most matching methods failed to detect the species complexes that were present in 1977. For the current dataset, there was moderate-high success in identifying specimens (53-99%). For both datasets, the capacity to discover new species was dependent on the methodological approach used. Species delimitation in New Zealand skinks was hindered by the absence of either a local or global barcoding gap, a result of recent speciation events and hybridisation. Whilst DNA barcoding is potentially useful for specimen identification and species discovery in New Zealand skinks, its error rate could hinder the progress of documenting biodiversity in this group. We suggest that integrated taxonomic approaches are more effective at discovering and describing biodiversity.     

Tracking the variability of phenotypic traits on a molecular phylogeny: an example from scolopendrid centipedes in peninsular India

Taxonomic studies on scolopendrid centipedes have often documented variability at the individual and population levels and applied those data to questions of species delimitation, but these investigations have mostly lacked an explicit phylogenetic framework. A molecular phylogeny and recent taxonomic revision for Indian species of the scolopendrid Digitipes Attems, 1930, permit variability of traditional taxonomic characters for Scolopendridae to be mapped onto a phylogeny. Based on their fit to the tree using maximum likelihood, reliable species-level characters include the number of glabrous antennal articles, presence of a median ridge on the tergites, and presence or absence of a tarsal spur on leg 20. Characters that are conserved within and diagnostic for particular species but labile within others (typically with geographic structure) include the first tergite with paramedian sutures, presence or absence of a lateral spine on the coxopleuron, and the number of spines in a ventromedial row on the ultimate leg prefemur. Comparisons with published accounts of variability in species of other scolopendrid genera, particularly Scolopendra and Otostigmus, show that Indian Digitipes has conserved morphology in some characters that are taxonomically useful elsewhere in the family, and most of its taxonomically informative characters have analogous patterns of variability in other genera. The approach used in this study to evaluate morphological variation in a phylogenetic framework can be applied to other taxa in which morphologically cryptic species have been reported and where species diagnosis requires a combination of characters.     

Excluding spatial sampling bias does not eliminate oversplitting in DNA‐based species delimitation analyses

DNA barcoding and DNA‐based species delimitation are major tools in DNA taxonomy. Sampling has been a central debate in this context, because the geographical composition of samples affects the accuracy and performance of DNA barcoding. Performance of complex DNA‐based species delimitation is to be tested under simpler conditions in absence of geographic sampling bias. Here, we present an empirical dataset sampled from a single locality in a Southeast‐Asian biodiversity hotspot (Laos: Phou Pan mountain). We investigate the performance of various species delimitation approaches on a megadiverse assemblage of herbivorous chafer beetles (Coleoptera: Scarabaeidae) to infer whether species delimitation suffers in the same way from exaggerate infraspecific variation despite the lack of geographic genetic variation that led to inconsistencies between entities from DNA‐based and morphology‐based species inference in previous studies. For this purpose, a 658 bp fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) was analyzed for a total of 186 individuals of 56 morphospecies. Tree‐based and distance‐based species delimitation methods were used. All approaches showed a rather limited match ratio (max. 77%) with morphospecies. Poisson tree process (PTP) and statistical parsimony network analysis (TCS) prevailingly over‐splitted morphospecies, while 3% clustering and Automatic Barcode Gap Discovery (ABGD) also lumped several species into one entity. ABGD revealed the highest congruence between molecular operational taxonomic units (MOTUs) and morphospecies. Disagreements between morphospecies and MOTUs have to be explained by historically acquired geographic genetic differentiation, incomplete lineage sorting, and hybridization. The study once again highlights how important morphology still is in order to correctly interpret the results of molecular species delimitation.     

A comparison of DNA‐based methods for delimiting species in a Cretan land snail radiation reveals shortcomings of exclusively molecular taxonomy

We compared the results of different approaches for delimiting species based on single‐locus DNA sequences with those of methods using binary multilocus data. As case study, we examined the radiation of the land snail genus Xerocrassa on Crete. Many of the methods based on mitochondrial sequences resulted in heavy under‐ or overestimations of the species number. The methods using AFLP data produced classifications with an on average higher concordance with the morphological classification than the methods based on mitochondrial sequences. However, the percentage of correct species classifications is low even with binary multilocus data. Gaussian clustering produced the classifications with the highest concordance with the morphological classification of all approaches applied in this study, both with single‐locus sequences and with binary multilocus data. There are two general problems that hamper species delimitation, namely rarity and the hierarchical structure of biodiversity. Methods for species delimitation using genetic data search for clusters of individuals, but do not implement criteria that are sufficient to distinguish clusters representing species from other clusters. The success of morphological species delimitation results from the potential to focus on characters that are directly involved in the speciation process, whereas molecular studies usually rely on markers that are not directly involved in speciation. © The Willi Hennig Society 2011.     

Integrative taxonomy of herbaceous plants with narrow fragmented distributions: A case study on Primula merrilliana species complex

An accurate understanding of species diversity is essential to studies across a wide range of biological subdisciplines. However, species delimitation remains challenging in evolutionary radiations, particularly in those herbaceous plants associated with microendemic, naturally fragmented distribution systems, where genotypic and phenotypic traits likely evolved discordantly. The Primula merrilliana complex, which is endemic to eastern China and has high horticultural value, used to be treated as one species but several clues suggested it might be composed of multiple species. Here we used multiple lines of evidence, including molecular, morphological, reproductive isolation, and geographic data, to assess independently evolving lineages within this complex. Our results indicated that the species diversity in the complex was underestimated previously, and four species (independently evolving lineages) can be recognized, including two new species described here. The extensive variation of the breeding system, especially the floral morph transition from distyled (outcrossing) to homostyled (selfing) multiple times, possibly promoted the rapid speciation within such a small geographic scale. This study case indicated that the phenomenon of genetically highly divergent but morphologically indistinguishable is perhaps shown in herbs with fragmented distributions; the alternative extreme evolutionary phenomenon, in which complete reproductive barriers have been accumulated but with little genetic differentiation, also exists. Thus we highlight the importance of incorporating other characters, such as postzygotic reproductive isolation and geographic data, with commonly used molecular and morphological traits to infer species boundaries through an integrative taxonomic approach in such systems.     

The African timber tree <Emphasis Type="Italic">Entandrophragma congoense</Emphasis> (Pierre ex De Wild.) A.Chev. is morphologically and genetically distinct from <Emphasis Type="Italic">Entandrophragma angolense</Emphasis> (Welw.) C.DC

Interpreting morphological variability in terms of species delimitation can be challenging. However, correcting species delineation can have strong implications for the sustainable management of exploited species. Up to now, species delimitation between two putative timber species from African forests, Entandrophragma congoense and E. angolense, remained unclear. To investigate their differences, we applied an integrated approach which combines morphological traits and genetic markers. We defined 13 morphological characters from 81 herbarium specimens and developed 15 new polymorphic microsatellite markers to genotype 305 samples (herbarium samples and specimens collected in the field across the species distribution ranges). Principal component analysis (PCA) of morphological data and the Bayesian clustering analyses of genetic data were used to assess differentiation between putative species. These analyses support two well-differentiated groups (F_ST?=?0.30) occurring locally in sympatry. Moreover, these two groups present distinct morphological characters at the level of the trunk, leaflets, and seeds. Our genetic markers identified few individuals (4%) that seem to be hybrids, though there is no evidence of genetic introgression from geographic patterns of genetic variation. Hence, our results provide clear support to recognize E. congoense as a species distinct from E. angolense, with a much lower genetic diversity than the latter, and that should be managed accordingly. This work highlights the power of microsatellite markers in resolving species boundaries.     

Species Delimitation and Analysis of Cryptic Species Diversity in the XXI Century

The potentials and limitations of different approaches to revealing species boundaries and describing cryptic species are discussed. Both the traditional methods of species delimitation, mostly based on morphological analysis, and the approaches using molecular markers are considered. Besides, the prospects of species identification using digital image recognition and machine learning are briefly considered. It is concluded that molecular markers provide very important material for species delimitation; the value of these data increases manifold if they can be compared with information on morphology, geographic distribution, and ecological preferences of the studied taxa. In many cases, only a practicing taxonomist who knows his or her group thoroughly can correctly interpret the molecular data and incorporate them into the existing knowledge system in order to make a taxonomic decision.

     

Species delimitation and digit number in a North African skink

Delimitation of species is an important and controversial area within evolutionary biology. Many species boundaries have been defined using morphological data. New genetic approaches now offer more objective evaluation and assessment of the reliability of morphological variation as an indicator that speciation has occurred. We examined geographic variation in morphology of the continuously distributed skink Chalcides mionecton from Morocco and used Bayesian analyses of nuclear and mitochondrial DNA (mtDNA) loci to examine: (i) their concordance with morphological patterns, (ii) support for species delimitation, (iii) timing of speciation, and (iv) levels of gene flow between species. Four digit individuals were found at sites between Cap Rhir (in the south) and the northern extreme of the range, whereas five‐digit individuals were found in two disjunct areas: (i) south of Cap Rhir and (ii) the north of the range where they were often syntopic with four‐digit individuals. The pattern of variation in generalized body dimensions was largely concordant with that in digit number, suggesting two general morphotypes. Bayesian analyses of population structure showed that individuals from sites south of Cap Rhir formed one genetic cluster, but that northern four‐ and five‐digit individuals clustered together. Statistical support for delimitation of these genetic clusters into two species was provided by a recent Bayesian method. Phylogenetic–coalescent dating with external time calibrations indicates that speciation was relatively recent, with a 95% posterior interval of 0.46–2.66 mya. This postdates equivalent phylogenetic dating estimates of sequence divergence by approximately 1 Ma. Statistical analyses of a small number of independent loci provide important insights into the history of the speciation process in C. mionecton and support delimitation of populations into two species with distributions that are spatially discordant with patterns of morphological variation.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

Evidence for high degrees of specialisation, evolutionary diversity, and morphological distinctiveness in the genus Bremia

Bremia lactucae is one of the most important pathogens in lettuce production. Recent molecular?studies revealed considerable genetic variation in this species complex. However, only few accessions from the same host have been examined for most species and no study investigating the morphological distinctiveness of phylogenetic lineages of Bremia has so far been reported. Thus it is believed that morphological species delimitation in Bremia is not feasible. In the present study, multiple accessions of neglected species, which had been described decades ago, but have not been widely accepted, were investigated, considering both multi-gene phylogenies and morphological characters. All previously described species from host genera other than Lactuca investigated, Bremia microspora, Bremia ovata, Bremia saussureae, and Bremia sonchicola, could be confirmed as distinct, host-specific entities. Also, morphological characteristics of their conidiophores and conidia allowed delimitation of these species. Therefore, not only the wide species concept to merge all Bremia species on the Asteraceae under B. lactucae is inappropriate but also their delimitation on the basis of morphological characters seems feasible. In addition, it has been shown that Bremia elliptica is phylogenetically distinct from the other species infecting the genus Lactuca, B. lactucae. It is therefore concluded that B. lactucae is most likely limited to Lactuca sativa and closely-related species, and that most species of Bremia are highly host specific. This finding might stimulate the search for durable resistance genes in genera closely related to the genus Lactuca and in divergent species of the genus itself.     

Compilation of morphological and molecular data,a necessity for taxonomy: The case of Hormogaster abbatissae sp. n. (Annelida,Clitellata, Hormogastridae)

Conflict among data sources can be frequent in evolutionary biology, especially in cases where one character set poses limitations to resolution. Earthworm taxonomy, for example, remains a challenge because of the limited number of morphological characters taxonomically valuable. An explanation to this may be morphological convergence due to adaptation to a homogeneous habitat, resulting in high degrees of homoplasy. This sometimes impedes clear morphological diagnosis of species. Combination of morphology with molecular techniques has recently aided taxonomy in many groups difficult to delimit morphologically. Here we apply an integrative approach by combining morphological and molecular data, including also some ecological features, to describe a new earthworm species in the family Hormogastridae, Hormogaster abbatissae sp. n., collected in Sant Joan de les Abadesses (Girona, Spain). Its anatomical and morphological characters are discussed in relation to the most similar Hormogastridae species, which are not the closest species in a phylogenetic analysis of molecular data. Species delimitation using the GMYC method and genetic divergences with the closest species are also considered. The information supplied by the morphological and molecular sources is contradictory, and thus we discuss issues with species delimitation in other similar situations. Decisions should be based on a profound knowledge of the morphology of the studied group but results from molecular analyses should also be considered.     

Cryptic Speciation Patterns in Iranian Rock Lizards Uncovered by Integrative Taxonomy

While traditionally species recognition has been based solely on morphological differences either typological or quantitative, several newly developed methods can be used for a more objective and integrative approach on species delimitation. This may be especially relevant when dealing with cryptic species or species complexes, where high overall resemblance between species is coupled with comparatively high morphological variation within populations. Rock lizards, genus Darevskia, are such an example, as many of its members offer few diagnostic morphological features. Herein, we use a combination of genetic, morphological and ecological criteria to delimit cryptic species within two species complexes, D. chlorogaster and D. defilippii, both distributed in northern Iran. Our analyses are based on molecular information from two nuclear and two mitochondrial genes, morphological data (15 morphometric, 16 meristic and four categorical characters) and eleven newly calculated spatial environmental predictors. The phylogeny inferred for Darevskia confirmed monophyly of each species complex, with each of them comprising several highly divergent clades, especially when compared to other congeners. We identified seven candidate species within each complex, of which three and four species were supported by Bayesian species delimitation within D. chlorogaster and D. defilippii, respectively. Trained with genetically determined clades, Ecological Niche Modeling provided additional support for these cryptic species. Especially those within the D. defilippii-complex exhibit well-differentiated niches. Due to overall morphological resemblance, in a first approach PCA with mixed variables only showed the separation between the two complexes. However, MANCOVA and subsequent Discriminant Analysis performed separately for both complexes allowed for distinction of the species when sample size was large enough, namely within the D. chlorogaster-complex. In conclusion, the results support four new species, which are described herein.