Delimitation Methods Research Articles

Let's get high: Cladogenesis in freshwater crabs (Decapoda: Potamonautidae: Potamonautes) supports the mountain gradient speciation hypothesis in the Cape Fold and Drakensberg Mountains, South Africa.

During the present study, the evolutionary relationship within a clade of mountain clade of freshwater crabs (Potamonautes) was examined using mtDNA sequence data for species from the Cape Fold Mountain (CFM) and Great Escarpment (Drakensberg Mountain range). We undertook phylogenetic analyses, divergence time estimation, and an ancestral area reconstruction to explore the period of cladogenesis and understand the biogeographic history in this high-altitude clade. Furthermore, we applied four species delimitation methods using ASAP, bPTP, bGMYC, and STACEY on the latter clade. Bayesian phylogenetic analyses retrieved a monophyletic freshwater crab clade comprised of two major sister clades, one comprised of the Cape Fold (clade A) and two comprised of Drakensberg Mountains (clade B) species. Divergence time estimation indicated that the two clades underwent Mio/Pliocene cladogenesis. Within the CFM clade (A), P. amathole (Amathola Mountains) was sister to P. parvispina (Cederberg and Kouebokkeveld Mountains) and the latter species were sister to P. parvicorpus (Cape Peninsula, Jonkershoek, and Helderberg Mountains) sister to P. tuerkayi (Overberg Mountains) and P. brincki (Hottentots Holland Mountains). Within the Drakensberg Mountain clade (B), we observed in situ diversification. Specimens from the southcentral Drakensberg Mountains (Dargle Forest, Injasuti, Karkloof, and Impendle) represent a new undescribed lineage Potamonautes sp. nov. 1. The second clade from the northern Drakensberg, representing P. clarus, was sister to a central Drakensberg Mountain clade that comprised P. depressus that was in turn sister to P. baziya from the Eastern Cape Province. The application of species delimitation methods generally overestimated the number of species. The biogeographic analyses indicated that the Eastern Cape Province is the most likely ancestral range area. Ecological niche modelling of representative species in clades A (Cape Fold Mountains) and B (Drakensberg Mountains) demonstrated that temperature and rainfall were the major abiotic drivers that differentiated the two clades. Our data favours the mountain gradient speciation hypothesis.

Distribution, diversity and diversification from DNA barcoding perspective: The case of Gammarus radiation in the ancient Lake Ohrid

AbstractAimA detailed, comparative DNA‐barcoding and morphospecies‐based overview of the vertical and horizontal distribution of Lake Ohrid's endemic Gammarus species flock is provided. Re‐evaluation of the time frame for the onset of the species flock and identification of events that putatively influenced diversification processes.LocationLake Ohrid, Balkan Peninsula, Europe.TaxonGammarus species flock (Amphipoda, Crustacea).MethodsExtensive sampling and DNA barcoding of 600 individuals was carried out. DNA sequences were analysed using species delimitation methods, haplotype network reconstructions, Bayesian molecular dating and demographic analyses. The COI‐based species delimitation results were validated with nuclear 28S RNA data.ResultsThe species flock distribution has a weak horizontal but clear vertical structure. The diversity along bathymetric gradients correlates with temperature and salinity; and the highest diversity is observed in sublittoral and springs on the lake's shore. Two new molecular units representing putatively new species are revealed and supported also by the nuclear marker. The onset of flock radiation overlaps with the time of lake formation. The COI gene shows signs of positive selection and an acceleration in substitution rate. The demographic changes within the flock happened during the last thousands years.Main ConclusionsDistribution of the Gammarus species flock is vertically structured, reflecting habitat zonation. Parapatric speciation is one of the possible mechanisms behind the flock's diversification. Detection of new MOTUs suggests that the flock's diversity is still not fully revealed. Simultaneously, failure to collect three previously known MOTUs suggests a loss of gammarid diversity in the lake. This represents, together with the current threats to the lake ecosystem (i.e. climate changes, development of tourism), a clear call for conservation efforts. The speciation events and demographic changes within the flock relate presumably to glacial and postglacial water level changes and to the colonisation of new depth ranges and the associated springs.

Mitochondrial genome structure and composition in 70 fishes: a key resource for fisheries management in the South Atlantic

BackgroundPhylogenetic gaps of public databases of reference sequences are a major obstacle for comparative genomics and management of marine resources, particularly in the Global South, where economically important fisheries and conservation flagship species often lack closely-related references. We applied target-enrichment to obtain complete mitochondrial genomes of marine ichthyofauna from the Brazilian coast selected based on economic significance, conservation status and lack of phylogenetically-close references. These included sardines (Dorosomatidae, Alosidae), mackerels (Scombridae) croakers (Sciaenidae), groupers (Epinephelidae) and snappers (Lutjanidae).ResultsCustom baits were designed to enrich mitochondrial DNA across a broad phylogenetic range of fishes. Sequencing generated approximately 100k reads per sample, which were assembled in a total of 70 complete mitochondrial genomes and include fifty-two new additions to GenBank, including five species with no previous mitochondrial data. Departures from the typical gene content and order occurred in only three taxa and mostly involved tRNA gene duplications. Start-codons for all genes, except Cytochrome C Oxidase subunit I (COI), were consistently ATG, whilst a wide range of stop-codons deviated from the prevailing TAA. Phylogenetic analysis confirmed assembly accuracy and revealed signs of cryptic diversification within the Mullus genus. Lineage delimitation methods using Sardinella aurita and S. brasiliensis mitochondrial genomes support a single Operational Taxonomic Unit.ConclusionsTarget enrichment was highly efficient, providing complete novel mitochondrial genomes with little sequencing effort. These sequences are deposited in public databases to enable subsequent studies in population genetics and adaptation of Latin American fish species and serve as a vital resource for conservation and management programs that rely on molecular data for species and genus-level identification.

Six new species of Margattea Shelford, 1911 (Blaberoidea, Pseudophyllodromiidae, Neoblattellini) from China.

Six Margattea species are established and described: three are cryptic species, namely, M.parabisignata Li & Che, sp. nov., M.semicircularis Li & Che, sp. nov., and M.forcipata Li & Che, sp. nov. They are distinguished from known species M.bisignata, M.spinifera, and M.paratransversa by their male genitalia with the aid of molecular species delimitation method (ABGD) using COI as the molecular marker. The other three new species are M.pedata Li & Che, sp. nov., M.undulata Li & Che, sp. nov., and M.bisphaerica Li & Che, sp. nov. Morphological and genitalia photographs of these new species of Margattea, as well as a key to the species of Margattea from China, are provided.

Barcoding Fails to Delimit Species in Mongolian Oedipodinae (Orthoptera, Acrididae).

Mongolia, a country in central Asia, with its vast grassland areas represents a hotspot for Orthoptera diversity, especially for the Acrididae. For Mongolia, 128 Acrididae species have been documented so far, of which 41 belong to the subfamily Oedipodinae (band-winged grasshoppers). Yet, few studies concerning the distribution and diversity of Oedipodinae have been conducted in this country. Molecular genetic data is almost completely absent, despite its value for species identification and discovery. Even, the simplest and most used data, DNA barcodes, so far have not been generated for the local fauna. Therefore, we generated the first DNA barcode data for Mongolian band-winged grasshoppers and investigated the resolution of this marker for species delimitation. We were able to assemble 105 DNA barcode (COI) sequences of 35 Oedipodinae species from Mongolia and adjacent countries. Based on this data, we reconstructed maximum likelihood and Bayesian inference phylogenies. We, furthermore, conducted automatic barcode gap discovery and used the Poisson tree process (PTP) for species delimitation. Some resolution was achieved at the tribe and genus level, but all delimitation methods failed to differentiate species by using the COI region. This lack of resolution may have multiple possible reasons, which likely differ between taxa: the lack of resolution in the Bryodemini may be partially explained by their massive genomes, implying the potential presence of large numbers of pseudogenes, while within the Sphingonotini incomplete lineage sorting and incorrect taxonomy are more likely explanations for the lack of signal. Further studies based on a larger number of gene fragments, including nuclear DNA, are needed to distinguish the species also at the molecular level.

Discovery of five new species of Allacta from Yunnan and Hainan, China (Blattodea, Pseudophyllodromiidae).

We examined new Allacta materials from Yunnan and Hainan Province, China, and discovered new species using both morphological and molecular species delimitation (ABGD) methods. Five new species are described: A.bifolium Li & Wang, sp. nov., A.hemiptera Li & Wang, sp. nov., A.lunulara Li & Wang, sp. nov., A.redacta Li & Wang, sp. nov., and A.unicaudata Li & Wang, sp. nov. All five species are placed under the hamifera species group. An updated key and checklist of Allacta species from China are provided.

Jump dispersal drives the relationship between micro- and macroevolutionary dynamics in the Sicydiinae (Gobiiformes: Oxudercidae) of Sundaland and Wallacea.

Insular biodiversity hotspots of Southeast Asia are remarkable for their biodiverse faunas. With a marine larval phase lasting up to several months, the freshwater fish subfamily Sicydiinae has colonized most islands of these hotspots. However, Sicydiinae diversity is still poorly understood in Southeast Asia. With the objective to estimate intraspecific genetic diversity and infer past demography, we conducted the molecular inventory of Sicydiinae species in Sundaland and Wallacea using 652bp of the mitochondrial cytochrome oxidase I gene, species delimitation methods and Bayesian Skyline plot reconstructions. In total, 24 Molecular Operational Taxonomic Units are delimited among the 603 sequences belonging to 27 species and five genera. Two cases of discordance between morphology and mitochondrial sequence are observed suggesting ongoing speciation and/or introgression in two genera. Multiple new occurrences are reported, either for a single biodiversity hotspot or both, some of which corresponding to observations of a few individuals far from the range distribution of their conspecifics. Among the ten species or species group whose intraspecific diversity was examined, high levels of genetic diversity and past population expansion are revealed by Tajima's D tests and Bayesian Skyline Plot reconstructions. Together these results indicate that long-distance dispersal is common and suggest that most endemic species originated through founder events followed by population expansion. Patterns of sexual dimorphism and males' coloration among diverging species pair seem to point to sexual selection as an important mechanism contributing to speciation in the Sicydiinae of Sundaland and Wallacea.

Variability in the white spot: a new genus and species of Discodorididae (Nudibranchia) from the central and western Pacific Ocean

ABSTRACT In this paper, a new genus in the nudibranch family Discodorididae, Avaldesia n. gen., is established for Avaldesia albomacula (Chan & Gosliner, 2007) and Avaldesia tahala (Chan & Gosliner, 2007), originally assigned to the genus Thordisa Bergh, 1877, and a new species, Avaldesia tamatoa n. sp., described here from the central Pacific. To establish species relationships within Avaldesia, as well as the placement of Avaldesia within Discodorididae, we utilized four molecular markers (cytochrome c oxidase subunit I, 16S rRNA, histone H3 and 28S rRNA) in our Bayesian inference and maximum likelihood analyses. Four species delimitation methods were complemented by morphological dissections and scanning electron microscopy. Our results reveal a clear separation between Avaldesia and Thordisa and suggest that Avaldesia is more closely related to the genera Hoplodoris Bergh, 1880 and Asteronotus Ehrenberg, 1831. The most characteristic features of Avaldesia include a radula with increasing denticulation towards the fimbriate outermost laterals and a reproductive system with a lobate vestibular gland, occasional hollow vestibular spine and a penis armed with one or more penial spines. All species of Avaldesia are found in shallow water (5–10 m depth) on rocky reefs, sandy sediments and algal fields with distributions across the Indo-Pacific.

Molecular diversity of Pseudoscorpiones in southern High Appalachian leaf litter.

The Pseudoscorpiones fauna of North America is diverse, but in regions like the southern Appalachian Mountains, they are still poorly documented with respect to their species diversity, distributions and ecology. Several families have been reported from these mountains and neighbouring areas. Here we analyse barcoding data of 136 specimens collected in leaf litter, most of them from high-elevation coniferous forest. We used ASAP as a species delimitation method to obtain an estimation of the number of species present in the region. For this and based on interspecific genetic distance values previously reported in Pseudoscorpions, we considered three different genetic Kimura two-parameter distance thresholds (3%/5%/8%), to produce more or less conservative estimates. These distance thresholds resulted in 64/47/27 distinct potential species representing the families Chthoniidae (33/22/12 species) and Neobisiidae (31/25/15) and at least six different genera within them. The diversity pattern seems to be affected by the Asheville Depression, a major biogeographic barrier in this area, with a higher diversity to the west of this geographic feature, particularly within the family Neobisiidae. The absence of representatives from other families amongst our studied samples may be explained by differences in their ecological requirements and occupation of different microhabitats.

Transcriptome data from silica-preserved leaf tissue reveal gene flow patterns in a Caribbean bromeliad.

Transcriptome sequencing is a cost-effective approach that allows researchers to study a broad range of questions. However, to preserve RNA for transcriptome sequencing, tissue is often kept in special conditions, such as immediate ultracold freezing. Here, we demonstrate that RNA can be obtained from 6-month-old, field-collected samples stored in silica gel at room temperature. Using these transcriptomes, we explore the evolutionary relationships of the genus Pitcairnia (Bromeliaceae) in the Dominican Republic and infer barriers to gene flow. We extracted RNA from silica-dried leaf tissue from 19 Pitcairnia individuals collected across the Dominican Republic. We used a series of macro- and micro-evolutionary approaches to examine the relationships and patterns of gene flow among individuals. We produced high-quality transcriptomes from silica-dried material and demonstrated that evolutionary relationships on the island match geography more closely than species delimitation methods. A population genetic examination indicates that a combination of ecological and geographical features presents barriers to gene flow in Pitcairnia. Transcriptomes can be obtained from silica-preserved tissue. The genetic diversity among Pitcairnia populations does not warrant classification as separate species, but the Dominican Republic contains several barriers to gene flow, notably the Cordillera Central mountain range.

Species Diagnosis and DNA Taxonomy.

The use of DNA has helped to improve and speed up species identification and delimitation. However, it also provides new challenges to taxonomists. Incongruence of outcome from various markers and delimitation methods, bias from sampling and skewed species distribution, implemented models, and the choice of methods/priors may mislead results and also may, in conclusion, increase elements of subjectivity in species taxonomy. The lack of direct diagnostic outcome from most contemporary molecular delimitation approaches and the need for a reference to existing and bestsampled trait reference systems reveal the need for refining the criteria of species diagnosis and diagnosability in the current framework of nomenclature codes and good practices to avoid nomenclatorial instability, parallel taxonomies, and consequently more and new taxonomic impediment.

Epitope delimitation: A new method for defining epitopes of human IgG-reactive antigenic peptides based on rabbit-recognized epitope motifs.

The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides.

Molecular diversity of Diplura in southern High Appalachian leaf litter.

The fauna of Diplura, the two-pronged bristletails (Hexapoda), of the southern Appalachians has received little focused systematic attention. Existing literature suggests the fauna to comprise around a dozen species. Based on a broader DNA barcode-based survey of high elevation litter arthropods in the region, we suggest the fauna to be much richer, with automated species delimitation methods hypothesising as many as 35 species, most highly restricted to single or closely proximate localities. Such a result should not be very surprising for such small, flightless arthropods, although it remains to be seen if other markers or morphology support such high diversity. The region still remains sparsely sampled for these more cryptic elements of the arthropod fauna and much larger numbers of species undoubtedly remain to be discovered.

An integrative taxonomic approach to the Atlantic Hansarsia (formerly Nematoscelis) yields new krill taxa (Crustacea: Euphausiidae)

A recent molecular phylogenetic and biogeographic study on the krill genus Hansarsia revealed undescribed cryptic diversity in the Atlantic. Each of four species analysed encompassed robust molecular clades that were linked to dimorphic males in H. microps, H. atlantica and H. tenella. We tested the robustness and divergence of the observed clades using an integrative approach including (1) three independent species delimitation methods for the mitochondrial COI gene (ASAP, GMYC, bPTP), (2) variability of two nuclear genes (H3 and ITS1) and (3) morphological analysis (MDS and PCA) with a dataset of 22 characters scored for 131 specimens. Both molecular and morphological analyses resulted in at least six distinct clades within the Atlantic Hansarsia. The strongest divergence was revealed between the two clades of H. tenella, one of which we diagnosed as a new species. Two clades of H. megalops also showed significant divergence but in the absence of males, we were reluctant to designate new species. Different clades linked to male forms in H. microps and H. atlantica are suggested as an incipient species. We also hypothesise an unusual trend in the evolution of euphausiids, in which visual recognition enhances tactile interaction during mating. Our results show that analyses of ostensibly well studied groups may yet yield taxonomic surprises. ZooBank: urn:lsid:zoobank.org:pub:AE045636-50EF-450A-B9B3-9231E8B91522

Delimiting Species with Single-Locus DNA Sequences.

DNA sequences are increasingly used for large-scale biodiversity inventories. Because these genetic data avoid the time-consuming initial sorting of specimens based on their phenotypic attributes, they have been recently incorporated into taxonomic workflows for overlooked and diverse taxa. Major statistical developments have accompanied this new practice, and several models have been proposed to delimit species with single-locus DNA sequences. However, proposed approaches to date make different assumptions regarding taxon lineage history, leading to strong discordance whenever comparisons are made among methods. Distance-based methods, such as Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP), rely on the detection of a barcode gap (i.e., the lack of overlap in the distributions of intraspecific and interspecific genetic distances) and the associated threshold in genetic distances. Network-based methods, as exemplified by the REfined Single Linkage (RESL) algorithm for the generation of Barcode Index Numbers (BINs), use connectivity statistics to hierarchically cluster-related haplotypes into molecular operational taxonomic units (MOTUs) which serve as species proxies. Tree-based methods, including Poisson Tree Processes (PTP) and the General Mixed Yule Coalescent (GMYC), fit statistical models to phylogenetic trees by maximum likelihood or Bayesian frameworks.Multiple webservers and stand-alone versions of these methods are now available, complicating decision-making regarding the most appropriate approach to use for a given taxon of interest. For instance, tree-based methods require an initial phylogenetic reconstruction, and multiple options are now available for this purpose such as RAxML and BEAST. Across all examined species delimitation methods, judicious parameter setting is paramount, as different model parameterizations can lead to differing conclusions. The objective of this chapter is to guide users step-by-step through all the procedures involved for each of these methods, while aggregating all necessary information required to conduct these analyses. The "Materials" section details how to prepare and format input files, including options to align sequences and conduct tree reconstruction with Maximum Likelihood and Bayesian inference. The Methods section presents the procedure and options available to conduct species delimitation analyses, including distance-, network-, and tree-based models. Finally, limits and future developments are discussed in the Notes section. Most importantly, species delimitation methods discussed herein are categorized based on five indicators: reliability, availability, scalability, understandability, and usability, all of which are fundamental properties needed for any approach to gain unanimous adoption within the DNA barcoding community moving forward.

Diaporthe species on palms – integrative taxonomic approach for species boundaries delimitation in the genus Diaporthe, with the description of D. pygmaeae sp. nov.

The application of traditional morphological and ecological species concepts to closely related, asexual fungal taxa is challenging due to the lack of distinctive morphological characters and frequent cosmopolitan and plurivorous behaviour. As a result, multilocus sequence analysis (MLSA) has become a powerful and widely used tool to recognise and delimit independent evolutionary lineages (IEL) in fungi. However, MLSA can mask discordances in individual gene trees and lead to misinterpretation of speciation events. This phenomenon has been extensively documented in Diaporthe, and species identifications in this genus remains an ongoing challenge. However, the accurate delimitation of Diaporthe species is critical as the genus encompasses several cosmopolitan pathogens that cause serious diseases on many economically important plant hosts. In this regard, following a survey of palm leaf spotting fungi in Lisbon, Portugal, Diaporthe species occurring on Arecaceae hosts were used as a case study to implement an integrative taxonomic approach for a reliable species identification in the genus. Molecular analyses based on the genealogical concordance phylogenetic species recognition (GCPSR) and DNA-based species delimitation methods revealed that speciation events in the genus have been highly overestimated. Most IEL identified by the GCPSR were also recognised by Poisson tree processes (PTP) coalescent-based methods, which indicated that phylogenetic lineages in Diaporthe are likely influenced by incomplete lineage sorting (ILS) and reticulation events. Furthermore, the recognition of genetic recombination signals and the evaluation of genetic variability based on sequence polymorphisms reinforced these hypotheses. New clues towards the intraspecific variation in the common loci used for phylogenetic inference of Diaporthe species are discussed. These results demonstrate that intraspecific variability has often been used as an indicator to introduce new species in Diaporthe, which has led to a proliferation of species names in the genus. Based on these data, 53 species are reduced to synonymy with 18 existing Diaporthe species, and a new species, D. pygmaeae, is introduced. Thirteen new plant host-fungus associations are reported, all of which represent new host family records for Arecaceae. This study has recognised and resolved a total of 14 valid Diaporthe species associated with Arecaceae hosts worldwide, some of which are associated with disease symptoms. This illustrates the need for more systematic research to examine the complex of Diaporthe taxa associated with palms and determine their potential pathogenicity. By implementing a more rational framework for future studies on species delimitation in Diaporthe, this study provides a solid foundation to stabilise the taxonomy of species in the genus. Guidelines for species recognition, definition and identification in Diaporthe are included.

Genetic and morphological evidence of a single species of bronze featherback (Notopterus notopterus) in Sundaland

The bronze featherback (Notopterus notopterus) is an iconic species of freshwater fish commonly found in South-East Asia and of great commercial and patrimonial importance. At present, the genus Notopterus comprises of two distinct species, Notopterus notopterus (Pallas, 1769) and Notopterus synurus (Bloch & Schneider 1801). On the Indonesian archipelago, genetic diversity and morphology of the bronze featherback were investigated for conservation purposes. The use of DNA-based species delimitation methods, applied to 165 Cytochrome oxidase I sequences of Notopteridae (121 belonging to N. notopterus), evidence a concordance between species and Molecular Operational Taxonomic Unit (MOTU) and the two species of Notopterus are recognized. In N. notopterus, 9 haplotypes are detected among the 121 sequences analyzed, and three are restricted to Sundaland. These three haplotypes had distinct geographic distribution with a haplotype observed in Java, Sumatra and Borneo, another haplotype restricted to South Sumatra and a third haplotype only found in Northern Sumatra. The analyze of 21 morphometric and 9 meristic variables revealed two groups within N. notopterus, which were only supported by subtle differences in measurements with overlapping distributions between groups. The present study supports the validity of Notopterus notopterus in Sundaland and the remarkable genetic continuity among populations across its range distribution.

Molecular and morphometric data provide evidence of intraspecific variation in shape and pigmentation pattern in Otocinclus cocama (Siluriformes: Loricariidae) across major river drainages.

Otocinclus cocama, a uniquely colored species of the loricariid catfish genus Otocinclus described solely from the type locality in the lower Ucayali River in northern Peru, is reported occurring in the Tigre River, a tributary to the Marañón River that drains a different section of the Andean Mountain range in the western Amazon. Both populations differ in the number of dark bars spanning the flanks of the body, and we investigated whether these morphotypes constitute distinct species. The body shapes of populations from the Tigre and Ucayali rivers were compared using geometric morphometrics. Although principal component analysis detected a broad overlap between populations, multivariate analysis of variance and linear driscriminat analysis revealed a subtle differentiation between the populations of the two hydrographic basins. Average body shape of the Ucayali River population tend to be slightly higher than that of the Tigre River, with the caudal peduncle stretched vertically in the Ucayali population. Multivariate regression of shape and centroid size revealed an allometric effect of 10.7% (p < 0.001), suggesting that the variation between Tigre and Ucayali populations was purely shape variation. Molecular data of coI, cytb, nd2, and 16S mitochondrial genes indicated a nucleotide diversity range from 0.001 to 0.003, and haplotypic diversity range from 0.600 ± 0.11 to 0.79 ± 0.07. The median-joining haplotype network for the concatenated matrix exhibited two divergent haplogroups related to the geographic area and separated by <10 mutational steps. The molecular species delimitation methods based on distance (automatic barcode gap discovery and assemble species by automatic partitioning) recovered two molecular lineages evolving independently, being one of the lineages formed by individuals from both populations. Tree-based methods (generalized mixed Yule coalescent and Bayesian implementation of the Poisson tree process) recovered similar topologies and supported single lineage recognition. Methods of molecular delimitation of species disclosed the high similarity between the two populations of Otocinclus cocama, further supported by the presence of old haplotypes common to both groups which could indicate that the populations still maintain gene flow. Although the morphological data reveal a subtle variation between both river basins, the molecular data suggest a weak population structuration based on hydrographic areas, but not different species lineages, therefore Otocinclus cocama is composed of a single lineage with two distinct morphotypes.

Limits of mitochondrial genes in delimiting species within a Carbula species complex (Hemiptera: Pentatomidae).

Molecular data has become a powerful tool for species delimitation, particularly among those that present limited morphological differences; while the mitochondrial genome, with its moderate length, low cost of sequencing and fast lineage sorting, has emerged as a practical data set. Due to the limited morphological differences among the closely related species of Carbula Stål 1865, the species boundaries between Carbula abbreviata (Motschulsky, 1866), Carbula humerigera (Uhler, 1860), and Carbula putoni (Jakovlev, 1876) have remained particularly unclear. In this study, we applied two phylogenetic reconstruction methods to two data sets (mitogenome and COI) to assess the phylogeny of Carbula distributed in Asia, and five species delimitation methods to determine the boundaries between East Asian Carbula species. Our phylogenetic analyses showed Carbula to be paraphyletic; the seven known species distributed within East Asia to form a single monophyletic group, and within this, C. abbreviata, C. humerigera, C. putoni and middle-type to comprise a C. humerigera species complex. Our results show that mitogenome data alone, while effective in the differentiation of more distantly related Carbula species, is not sufficient to accurately delimit the species within this newly described complex.

A systematic review of the Iberian springsnail subgenus Alzoniella (Navarriella) (Caenogastropoda: Hydrobiidae), with the description of a new potentially relict subfamily

Abstract The threatened springsnail subgenus Alzoniella (Navarriella) in the Iberian Peninsula has been suggested to be an old and relict lineage of the family Hydrobiidae. The subgenus is represented by two morphological species, both endemic to the Pyrenees and their southern foothills. We conducted phylogenetic analyses based on mitochondrial and nuclear gene fragments of topotypes and other populations, four molecular species delimitation methods, and morphological examinations to clarify the uncertain systematic position of the subgenus within the family, assess its species diversity, and understand the population genetic structure of the two geographically restricted species. Our phylogenetic results revealed that Alzoniella (Navarriella) is distantly related to all other species of Alzoniella, even belonging to an independent subfamily-level clade, for which we introduce the new genus Navarriella and the new subfamily Navarriellinae subfam. nov. Molecular methods and geometric morphometric analysis of shell shape identified a single species in the new genus. The significant phylogenetic distance from other hydrobiid taxa, narrow distribution, and limited gene flow among its populations (estimated from mitochondrial cytochrome c oxidase subunit I sequences) highlight Navarriella as an isolated lineage within the family that requires urgent conservation attention. Furthermore, our results cast a new light on the northern Iberian Mountains as a dispersal barrier for ancient spring lineages.