Species Delimitation Algorithms Research Articles

Species delimitation and phylogenetic relationships of the Prionospio complex (Annelida, Spionidae) in the Northeast Atlantic

AbstractThe Prionospio complex comprises the most diverse and complex group within the polychaete family Spionidae. The phylogenetic relationships within the group are still poorly understood, and the generic breakdown is unstable. In this study, we assessed the diversity, relationships, and distribution of species of the Prionospio complex occurring in Norwegian waters. We analysed mitochondrial genomes and nuclear ribosomal DNA assembled via whole‐genome shotgun sequencing, and Sanger sequenced fragments of COI and 16S rDNA. Sanger sequencing proved challenging in the group, where COI was only amplified successfully in 14% of specimens. By molecular species delimitation algorithms, our study revealed the presence of four well‐supported but currently undescribed species of Prionospio in Norwegian waters. We observed a novel distribution pattern of polychaetes in coastal waters, where certain species demonstrated distribution ranges spanning over 7000 km. Such wide distribution parallels patterns of deep‐sea Prionospio species, suggesting that factors beyond recent anthropogenic translocations are involved. Our analysis of 38 mitochondrial genomes and ribosomal nuclear DNA enabled us to hypothesise on the phylogenetic relationships of 14 species of the Prionospio complex. The analysis suggested that two characters previously used to designate genera: the beginning of the branchiae from chaetiger 3 and the presence of pinnules on the branchiae, might have evolved more than one time within the complex. We return Aurospio banyulensis to the genus Prionospio according to the diagnosis of Aurospio resulting tree where this species was nested among Prionospio species. Our findings provide new insights into the diversity and distribution patterns of Prionospio species and contribute to a better understanding of marine benthic biodiversity and the importance of taxonomic accuracy in conservation and management practices.

DNA barcoding of the genus Gampsocleis (Orthoptera, Tettigoniidae) from China.

DNA barcoding is a useful addition to the traditional morphology-based taxonomy. A ca. 650 bp fragment of the 5' end of mitochondrial cytochrome c oxidase subunit I (hereafter COI-5P) DNA barcoding was sued as a practical tool for Gampsocleis species identification. DNA barcodes from 889 specimens belonging to 8 putative Gampsocleis species was analyzed, including 687 newly generated DNA barcodes. These barcode sequences were clustered/grouped into Operational Taxonomic Units (OTUs) using the criteria of five algorithms, namely Barcode Index Number (BIN) System, Assemble Species by Automatic Partitioning (ASAP), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP). The Taxon ID Tree grouped sequences of morphospecies and almost all MOTUs in distinct nonoverlapping clusters. Both long- and short-winged Gampsocleis species are reciprocally monophyletic in the Taxon ID Tree. In BOLD, 889 barcode sequences are assigned to 17 BINs. The algorithms ASAP, jMOTU, bPTP and GMYC clustered the barcode sequences into 6, 13, 10, and 23 MOTUs, respectively. BIN, ASAP, and bPTP algorithm placed three long-winged species, G. sedakovii, G. sinensis and G. ussuriensis within the same MOTU. All species delimitation algorithms split two short-winged species,G. fletcheri and G. gratiosa into at least two MOTUs each, except for ASAP algorithm. More detailed molecular and morphological integrative studies are required to clarify the status of these MOTUs in the future.

New sand fly (Diptera, Psychodidae) records and COI DNA barcodes in the state of Maranhão, Eastern Amazon, Brazil

The sand fly fauna and the usefulness of the DNA barcoding fragment of the cytochrome c oxidase subunit I (COI) gene were accessed in a forest fragment in the municipality of Governador Newton Bello, state of Maranhão, Brazil. We performed entomological collections in three independent campaigns in May and October 2021, and January 2023. Sand flies were morphologically-identified and then DNA barcoded. Sequences were deposited and analyzed in the BOLD System Database, and various species delimitation algorithms, to assess whether DNA sequences merge into taxonomic units in accordance with nominal species. In total, 1,524 sand flies were collected, comprising 32 nominal species. Nyssomyia antunesi was the most abundant species (31.5 %), followed by Psychodopygus davisi (27 %). We reported for the first time in the state of Maranhão, the presence of Lutzomyia evangelistai, Lutzomyia sherlocki, Pressatia equatorialis, and Psathyromyia barrettoi. We amplified and analyzed 67 COI barcodes of 23 species, which were merged with conspecific sequences extracted from GenBank. The maximum intraspecific p distances ranged from 0.0 % to 14.74 %, while the distances to the nearest neighbor varied from 1.67 % to 13.64 %. The phylogenetic gene tree and species delimitation tools clustered sequences into well-supported clades/clusters for each nominal species, except for Pressatia choti/Pr. equatorialis, which have the lowest interspecific genetic distance (1.67 %). We sequenced for the first time COI barcodes of Brumptomyia brumpti, Evandromyia monstruosa, Micropygomyia rorotaensis, Micropygomyia pilosa, Pintomyia christenseni, Pintomyia pacae, Pr. equatorialis, Pa. barrettoi, and Psathyromyia hermanlenti, which will be useful for further molecular identification and classification proposals of Neotropical species. This study updated the current list of the sand fly fauna for the state of Maranhão to 97, and demonstrated that COI barcodes are useful for specific identification.

Molecular and morphometric study of Brazilian populations of Psychodopygus davisi.

In this study, we analysed the molecular and morphometric differences of several populations of the putative sand fly vector Psychodopygus davisi (Root, 1934) (Diptera, Psychodidae, Phlebotominae) in Brazil. We amplified the 658 base pair fragments of the DNA barcoding region-cytochrome c oxidase subunit 1 (COI) gene-for 57 specimens of P. davisi and three specimens of Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979). We merged our data with public sequences of the same species available from GenBank. Then, the combined dataset-87 sequences and 20 localities-was analysed using population structure analysis and different species delimitation approaches. Geometric morphometry of wings was performed for 155 specimens of P. davisi populations from the North, Midwest and Southeast Brazilian regions, analysing the differences in centroid sizes and canonical variates. Molecular analysis indicated high intraspecific genetic distance values for P. davisi (maximum p distance = 5.52%). All algorithms identified P. davisi and P. claustrei as distinct molecular taxonomic units, despite the low interspecific distance (p distance to the nearest neighbour = 4.79%). P. davisi sequences were split into four genetic clusters by population structure analysis and at least five genetic lineages using intermediate scenarios of the species delimitation algorithms. The species validation analysis of BPP strongly supported the five-species model in our dataset. We found high genetic diversity in this taxon, which is in agreement with its wide geographic distribution in Brazil. Furthermore, the wing analysis showed that specimens from the Southeast Region of Brazil are different from those in the North and the Midwest. The evolutionary patterns of P. davisi populations in Brazil suggest the presence of candidate species, which need to be validated in future studies using a more comprehensive approach with both genomic data and morphological characters.

Trichophoromyia auraensis: evidence for cryptic species and first record in the state of Maranhão, Brazil.

Trichophoromyia auraensis (Mangabeira, 1942) (Diptera, Psychodidae, Phlebotominae) has a wide geographic distribution in the western region of the Amazon biome, where it is a putative Leishmania vector. Here, we reported for the first time a population of this species in the Brazilian state of Maranhão, in the eastern Amazon, from which we DNA-barcoded and compared with previously processed specimens from Acre State, in the western Amazon. For this, we analyzed the DNA barcoding fragment (658 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) of Trichophoromyia species using phylogeneticgene trees, and species delimitation algorithms. The analyses of COI barcodes showed high values of genetic distance (mean K2P = 5.17) and well-supported clades/MOTUs for the eastern and western populations of T. auraensis, which may indicate a possible complex of cryptic species. The western population of this taxon merged with the close-related sand fly Trichophoromyia velezbernali Posada-López, Galvis and Galati, 2018 from Colombia, which may be associated with the recent speciation history and introgression between these populations. These evidences should be evaluated with a more comprehensive sampling in terms of analyzed populations and molecular markers.

Improving the COI DNA barcoding library for Neotropical phlebotomine sand flies (Diptera: Psychodidae)

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.Graphical

Ancistroceruswasps (Hymenoptera, Vespidae) from the Centre of Europe: Phylogeny, cryptic species, neutral and non‐neutral markers

AbstractThe aim of the study was to clarify the phylogenetic relationships among Northern EuropeanAncistrocerusand comparison of the applicability of evolutionarily neutral and non‐neutral markers for reconstruction of phylogeny. We used a 19,400 bp long dataset that included parts of mitochondrial DNA, nuclear rDNA operon, and 10 nuclear protein‐coding genes. Application of molecular barcoding and species delimitation algorithms unveiled a presence of cryptic species,A. balticussp. n., in the trap‐nesting wasp communities of the centre of Europe. We assessed the morphological, biological, and ecological differences of it from the siblingA. trifasciatusand updated the regional identification key. Phylogeny reconstruction using the neutral and the presumably non‐neutral markers resulted in different tree topologies. Evolutionary congruence of the rDNA operon with the other markers was relatively low. Evolutionary rate of the mitochondrial genes was 7–8 times as high as that of the exons of the nuclear genes, therefore, the mitochondrial markers overshadowed the nuclear ones in the phylogeny reconstructions. We assumed that at the speciation level, we might consider two different patterns of phylogeny: one based on evolutionary time and neutral changes, and the other based on adaptive evolutionary pathways under directional selection pressures. We assessed the effect of directional selection on the nuclear protein‐coding genes, applying the Spearman's rank correlation between pairwise phylogenetic distances among species, estimated using exons, and these distances, estimated using introns. One of these markers demonstrated a lack of positive correlation, implying a variable directional selection pressure on the coded protein. The publication has been registered on ZooBank: urn:lsid:zoobank.org:pub:13BD28D0‐736D‐4B2A‐B5CF‐4824BD4CDCFB.

DNA barcoding and morphology reveal European and western Asian Arctiavillica (Linnaeus, 1758) as a complex of species (Lepidoptera, Erebidae, Arctiinae).

Currently, the genus Arctia Schrank, 1802 includes approximately 16 species in the Palaearctic region, depending on the taxonomic interpretation. Here, populations of the Arctiavillica (Linnaeus, 1758) morphospecies complex were studied from Europe to the Middle East (Turkey, northern Iran) by molecular methods. Morphological treatment has traditionally revealed the presence of five nominal taxa: A.villica (Linnaeus, 1758), A.angelica (Boisduval, 1829), A.konewkaii (Freyer, 1831), A.marchandi de Freina, 1983, and A.confluens Romanoff, 1884. The molecular approach tests whether they represent well-delimited species. Subsequently, this study corroborates the suitability of the mitochondrial cytochrome c oxidase subunit 1 (COI) marker sequence for species delimitation. In total, 55 barcodes of the Arctiavillica complex were compared, and two molecular species delimitation algorithms were applied to reveal the potential Molecular Operational Taxonomic Units (MOTUs), namely the distance-based Barcode Index Number (BIN) System, and the hierarchical clustering algorithm based on a pairwise genetic distances approach using the Assemble Species by Automatic Partitioning (ASAP). The applied ASAP distance-based species delimitation method for the analysed dataset revealed an interspecific threshold of 2.0-3.5% K2P distance as suitable for species identification purposes of the Iberian A.angelica and the Sicilian A.konewkaii and less than 2% for the three taxa of the A.villica clade: A.villica, A.confluens, and A.marchandi. This study contributes to a better understanding of the taxonomy of the genus Arctia and challenges future revision of this genus in Turkey, the Caucasus, Transcaucasia as well as northern Iran using standard molecular markers.

DNA barcoding as a valuable tool for delimiting mollusk species of the genus Biomphalaria Preston, 1910 (Gastropoda: Planorbidae).

The genus Biomphalaria in Brazil includes 11 species and one subspecies, three of which are intermediate hosts of Schistosoma mansoni. Due to the recent evolution of this group, some species are difficult to identify based on morphological characters, making the use of genetic markers necessary for species identification. This study aimed to evaluate the use of partial sequences of the cytochrome c oxidase I (coi) gene for the identification of Biomphalaria species using phylogenetic reconstruction and species delimitation algorithms. The study tested the use of DNA barcoding technique for species delimitation within the genus. DNA barcoding was performed by sequencing a partial region of the coi gene from specimens, and the sequences were analyzed using phylogenetic reconstruction and algorithms to delimit Operational Taxonomic Units (OTUs). The study found that the use of the coi gene in the reconstruction of the phylogeny of the genus might be an alternative for understanding the evolution and dispersion of species. However, this marker alone is not enough to solve complex taxonomic problems within the genus. A total of 223 sequences were analyzed, 102 of which could be separated using the barcode gap, enabling the correct identification of seven taxa. The study demonstrated that accurate mollusk identification is necessary for effective schistosomiasis control. The DNA barcoding methodology was found to be promising for accurate mollusk identification, which is crucial for concentrating schistosomiasis control efforts in places where it is needed.

COI-Barcoding and Species Delimitation Assessment of Toad-Headed Agamas of the Genus Phrynocephalus (Agamidae, Squamata) Reveal Unrecognized Diversity in Central Eurasia

We provide a diversity assessment of the agamid genus Phrynocephalus Kaup, 1825. We analyze COI mtDNA barcodes from 385 individuals sampled all over Phrynocephalus range. We apply the ABGD, ASAP, bGMYC, mlPTP and hsPTP species delimitation algorithms to analyze the COI gene fragment variation and assess the species diversity in Phrynocephalus. Nine species groups are revealed in Phrynocephalus in agreement with earlier studies on the phylogenetic relationships of the genus. We demonstrate that the present taxonomy likely underestimates the actual diversity of the genus. Alternative species delimitation algorithms provide a confusingly wide range of possible number of Phrynocephalus species—from 54 to 103 MOTUs (molecular operational taxonomic units). The ASAP species delimitation scheme recognizing 63 MOTUs likely most closely fits the currently recognized taxonomic framework of Phrynocephalus. We also report on 13 previously unknown Phrynocephalus lineages as unverified candidate species. We demonstrate that the ASAP and the ABGD algorithms likely most closely reflect the actual diversity of Phrynocephalus, while the mlPTP and hsPTP largely overestimate it. We argue that species delimitation in these lizards based exclusively on mtDNA markers is insufficient, and call for further integrative taxonomic studies joining the data from morphology, mtDNA and nuDNA markers to fully stabilize the taxonomy of Phrynocephalus lizards.

DNA Barcoding of genus Culicoides biting midges (Diptera: Ceratopogonidae) in the Brazilian Amazon

Culicoides biting midges are capable to transmit Oropouche virus, Bluetongue virus and Mansonella spp. This study aimed to assess the utility of DNA barcode as an alternative method in the Culicoides species identification. The study was conducted in Jamari National Forest. Biting midges were collected using HP light traps during four months, February, April, August and October 2018. Insects were morphologically identified to the species level, and rest of the body were subjected to DNA extraction and PCR targeting a fragment of the cytochrome c oxidase subunit I (COI) gene, which were analyzed and deposited in GenBank. A phylogenetic gene tree was reconstructed using RAxML software, and the sequences were assigned at Molecular Operational Taxonomic Unit (MOTU) level by species delimitation algorithms. According to morphological approach, 18 species of 2 subgenera and 7 species groups were identified. A total of 191 new COI barcodes from 18 species were generated. Of these, fifteen species have been deposited for the first time in all datasets in the world. These sequences allowed the correct identification of 188 and 187 specimens according to the BM and BCM criteria, respectively. The intraspecific genetic distances ranged from 0 to 16.5%, while the interspecific ones ranged from 2.1 to 27.1%. The nominal species Culicoides glabellus and C. tetrathyris splitted into three and two MOTUs, respectively, except for mPTP, indicating a cryptic diversity in these species. Also, sequences of C. pseudodiabolicus formed two MOTUs using all algorithms, except for PTP and ABGD, suggesting the existence of two potential species. In contrast, some barcodes of C. quasiparaensis and C. paraensis merged into a single MOTU, which can be explained by the complex characteristics of the paraensis group, since these species have similar morphological characters. Here, we provided the first COI barcodes for biting midges in Rondônia and Brazil, and demonstrates that these are sufficient to discriminate between some species.

Rediscovery of Plasmodium (Huffia) huffi (Apicomplexa, Haemosporida): a lost lineage from toucans.

Literature data on toucans haemosporidians are scarce and all reports come from investigations in Brazil. Muniz et al. (Rev Bras Malariol 3: 339-356, Muniz et al., Rev Bras Malariol 3:339-356, 1951) and Muniz and Soares (Rev Bras Malar 611-617, Muniz J, Soares R de RL (1954) Nota sôbre um parasita do gênero Plasmodium encontrado no Ramphastos toco Müller, 1776, "Tucano-Açu", e diferente do Plasmodium huffi: Plasmodium pinottii n. sp. Rev Bras Malar 611 - 617.) described two Plasmodium species, P. huffi and P. pinottii, in Ramphastos toco. Later, Manwell and Sessler (J Protozol 18: 570-574, Manwell and Sessler, Malaria Parasites of Toucans J Protozol 18:570-574, 1971) established a new subspecies, P. nucleophilum toucani. In the last review on avian haemosporidians, Valkiūnas (Valkiūnas, Avian malaria parasites and other haemosporidia, CRC Press, New York, 2005) highlighted that P. huffi was insufficiently characterized, considering it a lost lineage. Also, the original description of P. huffi was considered insufficiently clear, due to a possible co-infection of the toucan hosts with a Novyella-like species. Here, we redescribed the species Plasmodium (Huffia) huffi based on morphological and molecular data, which were found in two toucan species, Ramphastos toco and Pteroglossus aracari from Brazil. Morphological features of the specimens are markedly the same as the original description. In R. toco, we observe two individuals infected, one infected only with P. huffi and one co-infected with P. huffi and the Novyella-like species, as observed in the original description. Also, we observe one R. toco infected only with the Novyella-like species, identified by morphological and molecular data as Plasmodium (Novyella) nucleophilum nucleophilum. In this way, it was possible to redescribe Plasmodium huffi in detail, without the doubt characters observed in the original description. Moreover, by applying species delimitation algorithms to a large Plasmodium phylogeny, we were able to identify new possible hosts for P. huffi and extend its geographic distribution to include North America.

“More than meets the eye”: phylogeographic inferences and remarkable cryptic diversity and in endemic catfish Parotocinclus (Loricariidae: Hypoptopomatinae) from neglected and impacted basins in South America

The restricted range, taxonomic uncertainties and the apparent phylopatric behavior of Parotocinclus (dwarf plecos) make this fish group an excellent model to infer diversification patterns in Neotropical rivers. Thus, the aim of this study was to investigate the spatial–temporal relationships and the presence of cryptic forms in Parotocinclus cristatus from poorly known and human-impacted drainages in northeastern Brazil based on mitochondrial COI (589 bp) and Cyt-b (835 bp) and nuclear (Rhodopsin, 473 bp) markers. The phylogenetic reconstruction, haplotype network and species delimitation algorithms revealed three conspicuous and restricted molecular operational units (MOTUs) within P. cristatus. The samples from Cachoeira and Una basins represent a single MOTU dating back to the Early Pliocene, resulting in deep genetic divergence when compared to the other lineages. The MOTUs from Contas and Almada river basins diverged around 2.1 Ma, while the latter encompassed highly structured populations between upper and lower watersheds (0.6 Ma, Pleistocene). The closest relationship between more distant basins than nearby drainages indicates an intricate scenario of headwater captures that challenge traditional studies based on current geographic reliefs and ichthyofauna inventories. Therefore, caution when biogeographic inferences are carried out without further understanding about the genetic diversity and the geomorphological history, as commonly reported in fishes from eastern basins in Brazil. Finally, the conspicuous Linnean and Wallacean shortfalls in dwarf plecos, including unique lineages from unprotected and highly degraded areas, justifies a thorough review of their taxonomic status since valid species are the major source of information for defining conservation policies.

The choices we make and the impacts they have: Machine learning and species delimitation in North American box turtles (Terrapene spp.).

Model-based approaches that attempt to delimit species are hampered by computational limitations as well as the unfortunate tendency by users to disregard algorithmic assumptions. Alternatives are clearly needed, and machine-learning (M-L) is attractive in this regard as it functions without the need to explicitly define a species concept. Unfortunately, its performance will vary according to which (of several) bioinformatic parameters are invoked. Herein, we gauge the effectiveness of M-L-based species-delimitation algorithms by parsing 64 variably-filtered versions of a ddRAD-derived SNP data set collected from North American box turtles (Terrapene spp.). Our filtering strategies included: (i) minor allele frequencies (MAF) of 5%, 3%, 1%, and 0% (= none), and (ii) maximum missing data per-individual/per-population at 25%, 50%, 75%, and 100% (= no filtering). We found that species-delimitation via unsupervised M-L impacted the signal-to-noise ratio in our data, as well as the discordance among resolved clades. The latter may also reflect biogeographic history, gene flow, incomplete lineage sorting, or combinations thereof (as corroborated from previously observed patterns of differential introgression). Our results substantiate M-L as a viable species-delimitation method, but also demonstrate how commonly observed patterns of phylogenetic discordance can seriously impact M-L-classification.

Molecular Characterization of Mosquito Diversity in the Balearic Islands.

Several outbreaks of mosquito-borne diseases have taken place in Europe in recent years. In Spain, both active and passive surveillance have demonstrated that dengue and West Nile viruses are currently circulating, and seven autochthonous dengue cases have been reported in the last 2 yr. The effectiveness of vector control programs largely depends on the accuracy of the taxonomic identification of the species. However, in Spain, identification almost completely relies on the use of morphological keys to characterize the mosquito fauna. This study investigates the congruence between molecular and morphological species boundaries in 13 Spanish mosquito taxa. The Cytochrome c oxidase subunit I (COI) gene region was sequenced from 60 adult specimens collected in Mallorca, plus several representatives from other Spanish regions for comparative purposes. Phylogenetic relationships were established using Bayesian and maximum-likelihood approaches. Using three species delimitation algorithms (ABGD, mPTP, and GMYC), we found strong evidence for cryptic speciation within Anopheles algeriensis Theobald, a widespread mosquito in the Mediterranean basin. We also delimited the Mallorcan rock pool mosquito Aedes mariae (Sergent & Sergent), from mainland European populations. Finally, we found difficulties in the use of wing characters in species keys to distinguish Culiseta annulata (Schrk) from Culiseta subochrea (Edwards). Given that these species are vectors of pathogens of medical relevance and have veterinary importance, their accurate taxonomic identification is essential in European vector surveillance programs.

Hidden biodiversity in Neotropical streams: DNA barcoding uncovers high endemicity of freshwater macroinvertebrates at small spatial scales.

Aquatic macroinvertebrates play a crucial role in freshwater ecosystems, but their diversity remains poorly known, particularly in the tropics. This “taxonomic void” limits our understanding of biodiversity patterns and processes in freshwater ecosystems, and the scale at which they operate. We used DNA barcoding to estimate lineage diversity (and the diversity of unique haplotypes) in 224 specimens of freshwater macroinvertebrates at a small spatial scale within the Panama Canal Watershed (PCW). In addition, we compiled available barcoding data to assess macroinvertebrate diversity at a broader spatial scale spanning the Isthmus of Panama. Consistently across two species delimitation algorithms (i.e., ABGD and GMYC), we found high lineage diversity within the PCW, with ~ 100–106 molecular operational taxonomic units (MOTUs) across 168 unique haplotypes. We also found a high lineage diversity along the Isthmus of Panama, but this diversity peaked within the PCW. However, our rarefaction/extrapolation approach showed that this diversity remains under-sampled. As expected, these results indicate that the diversity of Neotropical freshwater macroinvertebrates is higher than previously thought, with the possibility of high endemicity even at narrow spatial scales. Consistent with previous work on aquatic insects and other freshwater taxa in this region, geographic isolation is likely a main factor shaping these patterns of diversity. However, other factors such as habitat variability and perhaps local adaptation might be reshaping these patterns of diversity at a local scale. Although further research is needed to better understand the processes driving diversification in freshwater macroinvertebrates, we suggest that Neotropical streams hold a high proportion of hidden biodiversity. Understanding this diversity is crucial in the face of increasing human disturbance.

Intrageneric evolutionary timing and hidden genetic diversity of Paramecium lineages (Ciliophora: Oligohymenophorea)

Members of the genus Paramecium (Ciliophora, Oligohymenophorea) are widespread ciliates able to colonize many fresh, brackish and marine water environments around the globe. Despite they have been historically used in a variety of research fields, ranging from genomics to ecology, their evolutionary origin and genetic structure remains elusive for most valid species. There are currently 19 valid species distributed in 5 subgenera, which can have restricted to cosmopolitan distribution patterns. However, their true biodiversity and biographical distribution are blurred by evidences of cryptic species within most of these valid species. Here, we inferred a 18S rDNA-based time-calibrated tree to determine the evolutionary origin of extant Paramecium lineages and intra-genus phylogenetic relationships. Next, we used two common computational species delimitation algorithms (PTP and mPTP) to analyze a population-level (cytochrome oxidase subunit I [COI]) phylogeny to predict the existence of independent evolutionary unities (IEUs) within valid Paramecium morphospecies. Our data suggest that, some cosmopolitan Paramecium may have emerged prior to the Cretaceous period (before the complete separation of the continents), thus, explaining their wide distribution pattern. We found that the diversity of cryptic species may be higher than previous though and that most of the valid Paramecium species should be, in fact considered as complexes of species, composed each by IEUs that can co-occur within restricted or wide geographical areas. Moreover, our data indicate that these species delimitation algorithms (PTP and mPTP) may be of great assistance to uncover the diversity of cryptic species within Paramecium (or any other microeukaryote) by indicating potential candidates for more in detail future studies of taxonomists through integrative taxonomy frameworks.

Faecal DNA to the rescue: Shotgun sequencing of non-invasive samples reveals two subspecies of Southeast Asian primates to be Critically Endangered species

A significant number of Southeast Asian mammal species described in the 19th and 20th century were subsequently synonymized and are now considered subspecies. Many are affected by rapid habitat loss which creates an urgent need to re-assess the conservation status based on species boundaries established with molecular data. However, such data are lacking and difficult to obtain for many populations and subspecies. We document via a literature survey and empirical study how shotgun sequencing of faecal DNA is a still underutilized but powerful tool for accelerating such evaluations. We obtain 11 mitochondrial genomes for three subspecies in the langur genus Presbytis through shotgun sequencing of faecal DNA (P. femoralis femoralis, P. f. percura, P. siamensis cf. cana). The genomes support the resurrection of all three subspecies to species based on multiple species delimitation algorithms (PTP, ABGD, Objective Clustering) applied to a dataset covering 40 species and 43 subspecies of Asian colobines. For two of the newly recognized species (P. femoralis, P. percura), the results lead to an immediate change in IUCN status to Critically Endangered due to small population sizes and fragmented habitats. We conclude that faecal DNA should be more widely used for clarifying species boundaries in endangered mammals.

Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes, Loricariidae)

Integrative approaches are particularly useful to resolve taxonomic uncertainties in species-rich groups that have undergone explosive radiation, such as Hypostomini (suckermouth armored catfishes). This tribe encompasses the genera Hypostomus and Pterygoplichthys, but the actual number of species in each genus and the intergeneric interrelationships are confusing or unknown for some ecoregions in the Neotropics. Here, we analyzed three endemic species Hypostomus chrysostiktos, H. jaguar and H. unae from Northeastern Mata Atlântica in northeastern Brazil based on meristic characters, geometric morphometrics, chromosomal data, DNA barcode and species delimitation algorithms. The current taxonomic status of these catfishes is challenged. While H. unae and H. jaguar revealed a close evolutionary relationship similar to those observed within Hypostomus from other basins, H. chrysostiktos was herein more closely related to Pterygoplichthys, being invariably recovered as a highly distinctive taxonomic unit than to Hypostomus. Therefore, we recommend the reallocation of H. chrysostiktos as Pterygoplichthys chrysostiktos and a thorough systematic review of Pterygoplichthys, particularly focusing on putative convergence traits in relation to Hypostomus.

Cryptic Diversity, but to What Extent? Discordance Between Single-Locus Species Delimitation Methods Within Mainland Anoles (Squamata: Dactyloidae) of Northern Central America.

Single-locus molecular barcoding is a useful method for identifying overlooked and undescribed biodiversity, providing the groundwork for further systematic study and taxonomic investigation. A variety of methods for delimiting species from barcoding libraries have been developed and applied, allowing for rapid estimates of species diversity in a broad range of taxa. However, tree-based and distance-based analyses can infer different group assignments, potentially over- or underestimating the number of putative species groups. Here, we explore diversity of mainland species of anole lizards from the Chortís Block biogeographical province of northern Central America using a DNA barcoding approach, generating and analyzing cytochrome oxidase subunit I (COI) sequences for over 400 samples assignable to 33 of 38 (86.8%) native and one introduced mainland species. We subsequently tested the effects different models of nucleotide substitution, different species-delimitation algorithms, and reducing our dataset had on species delimitation estimates. We performed of two distance-based (ABGD, RESL) and three tree-based (bPTP, mPTP, GMYC) analyses on both the full dataset and a dataset consisting only of unique halotypes. From 34 nominal taxa, analyses of the full dataset recovered between 34 and 64 operational taxonomic units (OTUs), while analyses of the reduced dataset inferred between 36 and 59. Reassigning individuals to either mPTP-inferred or ABGD clustered (7.2% threshold) groups improved the detection of a barcoding gap across three different models of nucleotide substitution, removing overlap between intra- and interspecific distances. Our results highlight the underestimated diversity of mainland Chortís Block anoles, but the lack of congruence between analyses demonstrates the importance of considering multiple analytical methods when dealing with single-locus datasets. We recommend future studies consider the effects of different models of nucleotide substitution on proposed barcoding gaps, as well as the effect reducing a dataset to unique haplotypes may have on proposed diversity estimates.