SPdel: A pipeline to compare and visualize species delimitation methods for single-locus datasets.

An accurate species delimitation is critical for biological studies. In this context, the use of molecular techniques along with species delimitation methods would help to a rapid and accurate biodiversity assessment. The species delimitation methods cluster data sets of orthologous sequences in molecular operational taxonomic units (MOTU). In particular, the methods based on a single gene are easily integrated with the widely used DNA barcoding approach. We developed SPdel a user-friendly pipeline to integrate different single-gene species delimitation methods. SPdel is designed to calculate and compare MOTUs obtained by different species delimitation approaches. SPdel also outputs diverse ready-to-publish quality figures, that facilitate the interpretation of results. SPdel aims to help researchers use species delimitation methods that would improve biodiversity studies.

Comparison between p-distance and single-locus species delimitation models for delineating reproductively tested strains of pennate diatoms (Bacillariophyceae) using cox1, rbcL and ITS.

Several automated molecular methods have emerged for distinguishing eukaryote species based on DNA sequence data. However, there are knowledge gaps around which of these single-locus methods is more accurate for the identification of microalgal species, such as the highly diverse and ecologically relevant diatoms. We applied genetic divergence, Automatic Barcode Gap Discovery for primary species delimitation (ABGD), Assemble Species by Automatic Partitioning (ASAP), Statistical Parsimony Network Analysis (SPNA), Generalized Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP) using partial cox1, rbcL, 5.8S + ITS2, ITS1 + 5.8S + ITS2 markers to delineate species and compare to published polyphasic identification data (morphological features, phylogeny and sexual reproductive isolation) to test the resolution of these methods. ASAP, ABGD, SPNA and PTP models resolved species of Eunotia, Seminavis, Nitzschia, Sellaphora and Pseudo-nitzschia corresponding to previous polyphasic identification, including reproductive isolation studies. In most cases, these models identified diatom species in similar ways, regardless of sequence fragment length. GMYC model presented smallest number of results that agreed with previous published identification. Following the recommendations for proper use of each model presented in the present study, these models can be useful tools to identify cryptic or closely related species of diatoms, even when the datasets have relatively few sequences.

DNA barcoding of Lutzomyia longipalpis species complex (Diptera: Psychodidae), suggests the existence of 8 candidate species.

The sand fly Lutzomyia (L.) longipalpis has been implicated as the primary vector of Leishmania infantum, the causative agent of visceral leishmaniasis VL. In addition, it has been associated with atypical cutaneous leishmaniasis transmission in the Neotropic and Central America, respectively. The existence of a L. longipalpis complex species has been suggested with important implications for leishmaniasis epidemiology; however, the delimitation of species conforming it remains a topic of controversy. The DNA Barcoding Initiative based on cox1 sequence variation was used to identify the MOTUs in L. longipalpis including previously described L. pseudolongipalpis. The genetic variation was analyzed based on tree and distance methods. Fifty-five haplotypes were obtained from 103 sequences which were assigned to MOTUs, with a clear separation and a high correspondence of individuals to the groups. Maximum likelihood and Bayesian phylogenetic analysis showed eight MOTUs (100% bootstrap) with high genetic divergence (12.6%). Data obtained in the present study suggest that L. longipalpis complex consists of at least 8 lineages that may represent species. It would be desirable perform additional morphological and molecular analysis of L. longipalpis from Colosó (Caribbean ecoregion) considering that specimens from that area were grouped with L. pseudolongipalpis one of the complex species previously described from Venezuela, which has not been registered in Colombia.

Use of species delimitation approaches to tackle the cryptic diversity of an assemblage of high Andean butterflies (Lepidoptera: Papilionoidea).

Cryptic biological diversity has generated ambiguity in taxonomic and evolutionary studies. Single-locus methods and other approaches for species delimitation are useful for addressing this challenge, enabling the practical processing of large numbers of samples for identification and inventory purposes. This study analyzed an assemblage of high Andean butterflies using DNA barcoding and compared the identifications based on the current morphological taxonomy with three methods of species delimitation (automatic barcode gap discovery, generalized mixed Yule coalescent model, and Poisson tree processes). Sixteen potential cryptic species were recognized using these three methods, representing a net richness increase of 11.3% in the assemblage. A well-studied taxon of the genus Vanessa, which has a wide geographical distribution, appeared with the potential cryptic species that had a higher genetic differentiation at the local level than at the continental level. The analyses were useful for identifying the potential cryptic species in Pedaliodes and Forsterinaria complexes, which also show differentiation along altitudinal and latitudinal gradients. This genetic assessment of an entire assemblage of high Andean butterflies (Papilionoidea) provides baseline information for future research in a region characterized by high rates of endemism and population isolation.

Cryptic speciation in populations of the genus Aphyocharax (Characiformes: Characidae) from eastern Amazon coastal river drainages and surroundings revealed by single locus species delimitation methods

Recent studies in eastern Amazon coastal drainages and their surroundings have revealed new fish species that sometimes exhibit little morphological differentiation (cryptic species). Thus, we used a DNA-based species delimitation approach to test if populations showing the morphotype and typical character states of the Aphyocharax avary holotype correspond either to A. avary or A. brevicaudatus, two known species from the region, or if they form independent lineages, indicating cryptic speciation. WP and GMYC analyses recovered five lineages (species) in the ingroup, while a bPTP analysis delimited three lineages. ABGD analyses produced two possible results: one corroborating the WP and GMYC methods and another corroborating the bPTP method. All methods indicate undescribed cryptic species in the region and show variation from at least 1 to 4 species in the ingroup, depending on the approach, corroborating previous studies, and revealing this region as a possible hotspot for discovering undescribed fish species.(AU)

Estudos recentes nas drenagens costeiras da Amazônia oriental e seus arredores revelaram novas espécies de peixes que às vezes exibem pouca diferenciação morfológica (espécies crípticas). Assim, usamos uma abordagem de delimitação de espécies baseada em DNA para testar se as populações que apresentam o morfotipo e os estados de caráter típicos do holótipo Aphyocharax avary correspondem a A. avary ou A. brevicaudatus, duas espécies conhecidas da região, ou se formam linhagens independentes, indicando especiação críptica. As análises de WP e GMYC recuperaram cinco linhagens (espécies) no grupo interno, enquanto uma análise de bPTP delimitou três linhagens. As análises ABGD produziram dois resultados possíveis: um corroborando os métodos WP e GMYC e outro corroborando o método bPTP. Todos os métodos indicam espécies crípticas não descritas na região e apresentam variação de pelo menos uma a quatro espécies no grupo interno, dependendo da abordagem, corroborando estudos anteriores, e revelando esta região como um possível "hotspot" para descoberta de espécies de peixes não descritas.(AU)

Cryptic speciation in populations of the genus Aphyocharax (Characiformes: Characidae) from eastern Amazon coastal river drainages and surroundings revealed by single locus species delimitation methods

Recent studies in eastern Amazon coastal drainages and their surroundings have revealed new fish species that sometimes exhibit little morphological differentiation (cryptic species). Thus, we used a DNA-based species delimitation approach to test if populations showing the morphotype and typical character states of the Aphyocharax avary holotype correspond either to A. avary or A. brevicaudatus, two known species from the region, or if they form independent lineages, indicating cryptic speciation. WP and GMYC analyses recovered five lineages (species) in the ingroup, while a bPTP analysis delimited three lineages. ABGD analyses produced two possible results: one corroborating the WP and GMYC methods and another corroborating the bPTP method. All methods indicate undescribed cryptic species in the region and show variation from at least 1 to 4 species in the ingroup, depending on the approach, corroborating previous studies, and revealing this region as a possible hotspot for discovering undescribed fish species.(AU)

Estudos recentes nas drenagens costeiras da Amazônia oriental e seus arredores revelaram novas espécies de peixes que às vezes exibem pouca diferenciação morfológica (espécies crípticas). Assim, usamos uma abordagem de delimitação de espécies baseada em DNA para testar se as populações que apresentam o morfotipo e os estados de caráter típicos do holótipo Aphyocharax avary correspondem a A. avary ou A. brevicaudatus, duas espécies conhecidas da região, ou se formam linhagens independentes, indicando especiação críptica. As análises de WP e GMYC recuperaram cinco linhagens (espécies) no grupo interno, enquanto uma análise de bPTP delimitou três linhagens. As análises ABGD produziram dois resultados possíveis: um corroborando os métodos WP e GMYC e outro corroborando o método bPTP. Todos os métodos indicam espécies crípticas não descritas na região e apresentam variação de pelo menos uma a quatro espécies no grupo interno, dependendo da abordagem, corroborando estudos anteriores, e revelando esta região como um possível "hotspot" para descoberta de espécies de peixes não descritas.(AU)

Pristimantis in the Eastern Brazilian Amazon: DNA barcoding reveals underestimated diversity in a megadiverse genus.

The genus Pristimantis has the highest species diversity among all terrestrial vertebrates, with most species observed in the Andean region and the Guiana Shield. Even with the recent description of a new species, only P. latro, P. dundeei and P. zimmermanae occur in the south of the Amazon River. The lack of taxonomists specialized in the field leads to the propagation of dubious terminologies (e.g. Pristimantis sp1, Pristimantis sp2, P. aff. Fenestratus and P. gr. conspicillatus) or even misidentification of species, resulting in erroneous species distributions. In this study, we applied the Automatic Barcode Gap Discovery (ABGD) algorithm for the delimitation of candidate species and values of genetic distances using the mitochondrial marker Cytochrome Oxidase Subunit I (COI), proposed in the barcode methodology, where values greater than 10% are considered as indicative of different species. We found large genetic distances between P. latro and Pristimantis sp1 Unconfirmed Candidate Species - UCS1 (21%), and between P. altamazonicus and Pristimantis sp2 UCS2 (14%). The ABGD method recognized UCS1 and UCS2 as distinct species. Pristimantis sp. UCS1 and UCS2 in the east of the Brazilian Amazon are indicated as candidate species. We suggest greater sampling of Pristimantis sp. UCS1 and UCS2, integrating morphology and bioacoustics to solve the taxonomic status in the east of the Brazilian Amazon.

The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity.

A molecular taxonomic study of the bladed Bangiales of the South Eastern Pacific (coast of Chile) was undertaken based on sequence data of the mitochondrial COI and chloroplast rbcL for 193 specimens collected from Arica (18°S) in the north to South Patagonia (53°S) in the south. The results revealed for the first time that four genera, Porphyra, Pyropia, Fuscifolium and Wildemania were present in the region. Species delimitation was determined based on a combination of a General Mixed Yule Coalescence model (GMYC) and Automatic Barcode Gap Discovery (ABGD) coupled with detection of monophyly in tree reconstruction. The overall incongruence between the species delimitation methods within each gene was 29%. The GMYC method led to over-splitting groups, whereas the ABGD method had a tendency to lump groups. Taking a conservative approach to the number of putative species, at least 18 were recognized and, with the exception of the recently described Pyropia orbicularis, all were new to the Chilean flora. Porphyra and Pyropia were the most diverse genera with eight 'species' each, whereas only a 'single' species each was found for Fuscifolium and Wildemania. There was also evidence of recently diverging groups: Wildemania sp. was distinct but very closely related to W. amplissima from the Northern Hemisphere and raises questions in relation to such disjunct distributions. Pyropia orbicularis was very closely related to two other species, making species delimitation very difficult but provides evidence of an incipient speciation. The difference between the 'species' discovered and those previously reported for the region is discussed in relation to the difficulty of distinguishing species based on morphological identification.