DNA barcoding ferns in an unexplored tropical montane cloud forest area of southeast Oaxaca, Mexico

DNA barcoding can be useful for species identification and phylogenetic analysis, but its effectivity has not been verified in most neotropical cloud forest plants. We tested three plastid barcodes, rbcLa, matK, and trnH-psbA, in selected pteridophytes, a well-represented group in these forests, from a little-explored area in Oaxaca, Mexico, applying the CBOL criteria for barcoding. We used BLASTn, genetic distance, and monophyly tree-based analyses employing neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference methods. Universal primers for rbcLa and trnH-psbA were successfully amplified and bi-directionally sequenced, but matK could not be amplified for most species. rbcLa showed the highest species discrimination in BLASTn (66.67%). trnH-psbA exhibited higher significant interspecific divergence values than rbcL and rbcLa + trnH-psbA (two-sample sign test, P value < 2.2e−16). Using NJ and ML phylogenetic trees, monophyletic species were successfully resolved (100%), differing only in support values and displaying full agreement with the most recent fern classification. ML trees showed the highest mean support value (80.95%). trnH-psbA was the only barcode that could detect the Elaphoglossoideae subfamily. Species discrimination did not increase using rbcLa + trnH-psbA. rbcLa is useful for fern barcoding, trnH-psbA is most helpful for phylogenetic analyses, and matK may not work as a universal barcoding marker.

Two new species of Byrrhinus Motschulsky, 1858 (Coleoptera, Limnichidae, Limnichinae) from Negros, Philippines

Two new species of Limnichidae beetles, Byrrhinus negrosensissp. nov. and Byrrhinus villarinisp. nov., are described from the Island of Negros in the Philippines. The adult specimens of the new species can be differentiated by patterns of body punctation, colour and orientation of elytral pubescence, posterolateral angle of pronotum, tarsomere length ratio and aedeagal form. Two clades, representing the two new species, were retrieved in the Maximum Likelihood gene tree using the 3’-end of the COI gene. Maximum genetic divergence within B. negrosensis sp. nov. and B. villarini sp. nov. were recorded to be 2.3% and 1.3%, respectively, while the mean interspecific divergence between the two new species was 19.7%. Morphological descriptions, digital photographs and COI sequences were provided for the two species. The state of knowledge of Byrrhinus is reviewed and an updated Philippine checklist is provided. By coupling morphological and molecular data, this paper provides the first additional new species of Philippine Byrrhinus in the last 28 years.

Genetic Diversity and Phylogeny of the Genus Euplotes (Protozoa, Ciliophora) Revealed by the Mitochondrial CO1 and Nuclear Ribosomal Genes

Nuclear ribosomal and mitochondrial genes have been utilized individually or in combination to identify known species and discriminate closely related species. However, compared with metazoans, genetic diversity within the ciliate order Euplotida is poorly known. The aim of this study is to investigate how much nucleotide sequence divergence occurs within Euplotes. A total of 14 new gene sequences, comprising four SSU rDNA and 10 CO1 (including three species for the first time) were obtained. Phylogenetic analyses were carried out based on sequences of two DNA fragments from the same 27 isolates. We found that CO1 revealed a larger interspecific divergence than the SSU rRNA gene, thus demonstrating a higher resolution for separating congeners. Genetic distances differ significantly at the species level. Euplotes balteatus was revealed to have a large intraspecific variation at two loci, while E. vannus showed different levels of haplotype variability, which appeared as a polyphyletic cluster on the CO1 tree. These high genetic divergences suggest the presence of more cryptic species. By contrast, the CO1 gene showed low variability within E. raikovi, appearing as monophyletic clusters, which indicates that this species could be identified based on this gene. Conclusively, CO1 is a suitable marker for the study of genetic diversity within Euplotes, and increased taxon sampling gives an opportunity to screen relationships among members of this genus. Additionally, current data present no clear biogeographical pattern for Euplotes.

Interspecific divergence of circadian properties in duckweed plants

The circadian clock system is widely conserved in plants; however, divergence in circadian rhythm properties is poorly understood. We conducted a comparative analysis of the circadian properties of closely related duckweed species. Using a particle bombardment method, a circadian bioluminescent reporter was introduced into duckweed plants. We measured bioluminescence circadian rhythms of eight species of the genus Lemna and seven species of the genus Wolffiella at various temperatures (20, 25, and 30 °C) and light conditions (constant light or constant dark). Wolffiella species inhabit relatively warm areas and lack some tissues/organs found in Lemna species. Lemna species tended to show robust bioluminescence circadian rhythms under all conditions, while Wolffiella species showed lower rhythm stability, especially at higher temperatures. For Lemna, two species (L. valdiviana and L. minuta) forming a clade showed relatively lower circadian stability. For Wolffiella, two species (W. hyalina and W. repanda) forming a clade showed extremely long period lengths. The circadian properties of species primarily reflect their phylogenetic positions. The relationships between geographical and morphological factors and circadian properties are also suggested.

Genome evolution of the psammophyte Pugionium for desert adaptation and further speciation

Deserts exert strong selection pressures on plants, but the underlying genomic drivers of ecological adaptation and subsequent speciation remain largely unknown. Here, we generated de novo genome assemblies and conducted population genomic analyses of the psammophytic genus Pugionium (Brassicaceae). Our results indicated that this bispecific genus had undergone an allopolyploid event, and the two parental genomes were derived from two ancestral lineages with different chromosome numbers and structures. The postpolyploid expansion of gene families related to abiotic stress responses and lignin biosynthesis facilitated environmental adaptations of the genus to desert habitats. Population genomic analyses of both species further revealed their recent divergence with continuous gene flow, and the most divergent regions were found to be centered on three highly structurally reshuffled chromosomes. Genes under selection in these regions, which were mainly located in one of the two subgenomes, contributed greatly to the interspecific divergence in microhabitat adaptation.

Atyid shrimps of the genus Caridina (Decapoda, Caridea, Atyidae) of Taipa-Coloane Island, Macau, China, with description of a new species

In this study, four species of atyid shrimps of the genus Caridina, viz., Caridina cantonensis Yü, 1938, C. breviata N. K. Ng & Cai, 2000, C. gracilipes De Man, 1892 and C. macauensis sp. nov., are documented from Macau, China for the first time. The new species, C. macauensis sp. nov., is identified using morphological and molecular data. According to its morphology, C. macauensis is distinguished from the closely related species C. trifasciata Yam & Cai, 2003, by a shorter rostrum, male second pereiopod and colouration. A diagnosis of the three known species and a full description of C. macauensis sp. nov. are presented. Furthermore, genetic data of C. macauensis sp. nov. derived from the mitochondrial cytochrome c oxidase subunit I (COI) support that this species has a sufficient interspecific divergence from its congeners.

Molecular phylogeny of the Anopheles hyrcanus group (Diptera: Culicidae) based on rDNA–ITS2 and mtDNA–COII

Background The Anopheles hyrcanus group, which includes 25 species, is widely distributed in the Oriental and Palaearctic regions. Given the difficulty in identifying cryptic or sibling species based on their morphological characteristics, molecular identification is regarded as an important complementary approach to traditional morphological taxonomy. The aim of this study was to reconstruct the phylogeny of the Hyrcanus group using DNA barcoding markers in order to determine the phylogenetic correlations of closely related taxa and to compare these markers in terms of identification efficiency and genetic divergence among species. Methods Based on data extracted from the GenBank database and data from the present study, we used 399 rDNA–ITS2 sequences of 19 species and 392 mtDNA–COII sequences of 14 species to reconstruct the molecular phylogeny of the Hyrcanus group across its worldwide range. We also compared the performance of rDNA–ITS2 against that of mtDNA–COII to assess the genetic divergence of closely related species within the Hyrcanus group. Results Average interspecific divergence for the rDNA–ITS2 sequence (0.376) was 125-fold higher than the average intraspecies divergence (0.003), and average interspecific divergence for the mtDNA–COII sequence (0.055) was eightfold higher than the average intraspecies divergence (0.007). The barcoding gap ranged from 0.015 to 0.073 for rDNA–ITS2, and from 0.017 to 0.025 for mtDNA–COII. Two sets of closely related species, namely, Anophels lesteri and An. paraliae, and An. sinensis, An. belenrae and An. kleini, were resolved by rDNA–ITS2. In contrast, the relationship of An. sinensis/An. belenrae/An. kleini was poorly defined in the COII tree. The neutrality test and mismatch distribution revealed that An. peditaeniatus, An. hyrcanus, An. sinensis and An. lesteri were likely to undergo hitchhiking or population expansion in accordance with both markers. In addition, the population of an important vivax malaria vector, An. sinensis, has experienced an expansion after a bottleneck in northern and southern Laos. Conclusions The topology of the Hyrcanus group rDNA–ITS2 and mtDNA–COII trees conformed to the morphology-based taxonomy for species classification rather than for that for subgroup division. rDNA–ITS2 is considered to be a more reliable diagnostic tool than mtDNA–COII in terms of investigating the phylogenetic correlation between closely related mosquito species in the Hyrcanus group. Moreover, the population expansion of an important vivax malaria vector, An. sinensis, has underlined a potential risk of malaria transmission in northern and southern Laos. This study contributes to the molecular identification of the Anopheles hyrcanus group in vector surveillance. Graphical abstract

Taxonomic revision of the genus Elmomorphus Sharp, 1888 I. Japanese and Korean species (Coleoptera: Dryopidae)

The species of Elmomorphus Sharp, 1888 occurring in Japan and Korea are redescribed and illustrated: E. brevicornis Sharp, 1888 (Japan, Korea) and E. amamiensis Nomura, 1959 (Japan). The standard barcoding fragment of the mitochondrial gene coding for cytochrome c oxidase subunit I (COI) was sequenced and used together with morphological characters to delimit the taxonomic boundaries of the two species. To assess their morphometric variation, eight morphometric characters were measured and statistically evaluated using principal component analysis. The two species of Elmomorphus formed distinct and well-separated clusters in the COI tree. Their interspecific divergence is very high, ranging from 22.7 to 23.9%. On the other hand, morphometric characters, including those previously presumed to be diagnostic, overlap and per se do not allow unambiguous species identification. Reliable morphological distinguishing characters are described for males and females. Molecular data along with the morphological evidence strongly confirm the species status of E. amamiensis. An identification key to the Japanese and Korean species is provided.

The queen conch mitogenome: intra- and interspecific mitogenomic variability in Strombidae and phylogenetic considerations within the Hypsogastropoda

Aliger gigas is an economically important and vulnerable marine species. We present a new mitogenome of A. gigas from the Mexican Caribbean and use the eight publicly available Strombidae mitogenomes to analyze intra- and interspecific variation. We present the most complete phylogenomic understanding of Hypsogastropoda to date (17 superfamilies, 39 families, 85 genera, 109 species) to revisit the phylogenetic position of the Stromboidea and evaluate divergence times throughout the phylogeny. The A. gigas mitogenome comprises 15,460 bp including 13 PCGs, 22 tRNAs, and two rRNAs. Nucleotide diversity suggested divergence between the Mexican and Colombian lineages of A. gigas. Interspecific divergence showed high differentiation among Strombidae species and demonstrated a close relationship between A. gigas and Strombus pugilis, between Lambis lambis and Harpago chiragra, and among Tridentarius dentatus/Laevistrombus canarium/Ministrombus variabilis. At the intraspecific level, the gene showing the highest differentiation is ATP8 and the lowest is NAD4L, whereas at the interspecific level the NAD genes show the highest variation and the COX genes the lowest. Phylogenomic analyses confirm that Stromboidea belongs in the non-Latrogastropoda clade and includes Xenophoridea. The phylogenomic position of other superfamilies, including those of previously uncertain affiliation, is also discussed. Finally, our data indicated that Stromboidea diverged into two principal clades in the early Cretaceous while Strombidae diversified in the Paleocene, and lineage diversification within A. gigas took place in the Pleistocene.

Integrative species delimitation based on COI, ITS, and morphological evidence illustrates a unique evolutionary history of the genus Paracercion (Odonata: Coenagrionidae)

Paracercion are common ‘blue and black’ colored damselflies. We explore the species boundaries of Paracercion (Odonata: Coenagrionidae) using ABGD, bPTP, GMYC and Distance-based clustering. We finally got the molecular data of all nine species of Paracercion. P. hieroglyphicum and P. melanotum were combined into one putative species based on cytochrome c oxidase I (COI). However, they were separated into two putative species based on the nuclear segment including ITS1-5.8S-ITS2 (ITS). This suggests the introgression of mtDNA in Paracercion. Paracercion barbatum and Paracercion melanotum can be separated into two species based on COI, whereas they were combined into one putative species based on ITS, which suggests a hybridization event between them. The lower interspecific divergence (COI: 0.49%) between P. barbatum and Paracercion v-nigrum indicates a recent speciation event in Paracercion. Paracercion sieboldii and P. v-nigrum can be separated into two putative species based on COI, while they were frequently merged into the same putative species based on ITS. This can be explained by incomplete lineage sorting in nDNA. Besides, P. pendulum and P. malayanum were synonymized as junior synonyms of P. melanotum. P. luzonicum was confirmed not to belong to Paracercion. The possibility of introgression, hybridization, recent speciation and incomplete lineage sorting makes species delimitation, based on molecular data, difficult and complicates understanding of the evolutionary history of Paracercion. The discordance in COI and ITS also indicates the value of using markers from different sources in species delimitation studies.