Paraphyletic Relationship Research Articles

Cicada minimum age tree: Cryptic speciation and exponentially increasing base substitution rates in recent geologic time

We developed a new time-calibrated tree incorporating primarily endemic along with some cryptic Ryukyu islands cicada data, following the recent publication of global cicada data by Marshall et al. (2018), Łukasik et al. (2018), Simon et al. (2019), Price et al. (2019), and Hill et al. (2021). A total of 352 specimens were analyzed using BEAST v1. X software with a relaxed clock model. Fossil calibrations as old as Triassic were adopted largely following Johnson et al. (2018) and Moulds (2018), and a Quaternary geological event calibration was adopted following Osozawa et al. (2012, 2021b) and input into BEAST v1. X. Our timetree suggests that Tettigarctidae had a cicada basal lineage as old as 200.63 Ma, with Derotettiginae the next oldest lineage at 99.2 Ma. Tibicininae is a sister of the remaining subfamilies of Tettigomyiinae, Cicadettinae, and Cicadidae, and their species level differentiation and radiation began at 40.57 Ma. The Cicadinae clade consists of specific tribes with paraphyletic relationship, and the vicariance and adaptive radiation generated many cryptic species in each tribe. We estimated base substitution rate as a function of age, and the result strongly indicates an exponential increase of base substitution rate in recent geologic time. The consequent increase in cicada biodiversity, including generation of cryptic species in the Ryukyu Islands and surroundings, may have been driven by the generation and spreading of C4 grasses and coeval Quaternary climate change.

Cicada minimum age tree: Cryptic speciation and exponentially increasing base substitution rates in recent geologic time

We developed a new time-calibrated tree incorporating primarily endemic along with some cryptic Ryukyu islands cicada data, following the recent publication of global cicada data by Marshall et al. (2018), Łukasik et al. (2018), Simon et al. (2019), Price et al. (2019), and Hill et al. (2021). A total of 352 specimens were analyzed using BEAST v1. X software with a relaxed clock model. Fossil calibrations as old as Triassic were adopted largely following Johnson et al. (2018) and Moulds (2018), and a Quaternary geological event calibration was adopted following Osozawa et al. (2012, 2021b) and input into BEAST v1. X. Our timetree suggests that Tettigarctidae had a cicada basal lineage as old as 200.63 Ma, with Derotettiginae the next oldest lineage at 99.2 Ma. Tibicininae is a sister of the remaining subfamilies of Tettigomyiinae, Cicadettinae, and Cicadidae, and their species level differentiation and radiation began at 40.57 Ma. The Cicadinae clade consists of specific tribes with paraphyletic relationship, and the vicariance and adaptive radiation generated many cryptic species in each tribe. We estimated base substitution rate as a function of age, and the result strongly indicates an exponential increase of base substitution rate in recent geologic time. The consequent increase in cicada biodiversity, including generation of cryptic species in the Ryukyu Islands and surroundings, may have been driven by the generation and spreading of C4 grasses and coeval Quaternary climate change.

Comparative genomics of the genus Halioglobus reveals the genetic basis for the reclassification of Halioglobus pacificus as Parahalioglobus pacificus gen. nov. comb. nov.

The genus Halioglobus is one of the environmentally relevant members of the family Halieaceae, class Gammaproteobacteria. At present, the genus is composed of three validly published species. However, in the recent study of the family Halieaceae, the species Halioglobus pacificus was observed to branch outside of the main clade formed by the members of Halioglobus, suggesting its distinct taxonomic placement within the family. In the present study, the taxonomic placement of H. pacificus was reassessed using comparative genomics. Phylogenomic analysis revealed the paraphyletic relationship of H. pacificus with the type species of the genus Halioglobus, and further demonstrated its genus-level placement. This phylogenetic relationship was reinforced by the average nucleotide and amino acid identity values shared by H. pacificus with the members of the family Halieaceae. Moreover, the results of the pan-genome analysis, together with the phenotype data, further supported the exclusion of H. pacificus from the genus Halioglobus. Based on these findings, the species H. pacificus is thereby assigned to a new genus Parahalioglobus gen. nov. as Parahalioglobus pacificus comb. nov.

Comparatively analyzing of chloroplast genome and new insights into phylogenetic relationships regarding the genus Stephania

The medicinal plant of the genus Stephania holds significant economic importance in the pharmaceutical industry. However, accurately classifying and subdividing this genus remains a challenge. Herein, the chloroplast (cp) genomes of Stephania and Cyclea were sequenced, and the primary characteristics, repeat sequences, inverted repeats regions, simple sequence repeats, and codon usage bias of 17 species were comparatively analyzed. Twelve markers were identified through genome alignment and sliding window analysis. Moreover, a molecular clock analysis revealed the divergence between subgenus (subg.) Botryodiscia and the combined Cyclea, subg. Stephania and Tuberiphania during the early Oligocene epoch. Notably, the raceme-type inflorescence represents the ancestral state of the Stephania and Cyclea. The genetic relationships inferred from the cp genome and protein-coding genes exhibited similar topologies. Additionally, the paraphyletic relationship between the genera Cyclea and Stephania was confirmed. Bayesian inference, maximum likelihood, and neighbor-joining trees consistently showed that section Tuberiphania and Transcostula were non-monophyletic. In conclusion, this research provides valuable insights for further investigations into species identification, evolution, and phylogenetics within the Stephania genus.

Complete chloroplast genome assembly and phylogenetic analysis of blackcurrant (Ribes nigrum), red and white currant (Ribes rubrum), and gooseberry (Ribes uva-crispa) provide new insights into the phylogeny of Grossulariaceae.

Blackcurrant (Ribes nigrum), red currant (R. rubrum), white currant (R. rubrum), and gooseberry (R. uva-crispa) belong to Grossulariaceae and are popular small-berry crops worldwide. The lack of genomic data has severely limited their systematic classification and molecular breeding. The complete chloroplast (cp) genomes of these four taxa were assembled for the first time using MGI-DNBSEQ reads, and their genome structures, repeat elements and protein-coding genes were annotated. By genomic comparison of the present four and previous released five Ribes cp genomes, the genomic variations were identified. By phylogenetic analysis based on maximum-likelihood and Bayesian methods, the phylogeny of Grossulariaceae and the infrageneric relationships of the Ribes were revealed. The four cp genomes have lengths ranging from 157,450 to 157,802 bp and 131 shared genes. A total of 3,322 SNPs and 485 Indels were identified from the nine released Ribes cp genomes. Red currant and white currant have 100% identical cp genomes partially supporting the hypothesis that white currant (R. rubrum) is a fruit color variant of red currant (R. rubrum). The most polymorphic genic and intergenic region is ycf1 and trnT-psbD, respectively. The phylogenetic analysis demonstrated the monophyly of Grossulariaceae in Saxifragales and the paraphyletic relationship between Saxifragaceae and Grossulariaceae. Notably, the Grossularia subgenus is well nested within the Ribes subgenus and shows a paraphyletic relationship with the co-ancestor of Calobotrya and Coreosma sections, which challenges the dichotomous subclassification of the Ribes genus based on morphology (subgenus Ribes and subgenus Grossularia). These data, results, and insights lay a foundation for the phylogenetic research and breeding of Ribes species.

Systematic studies on some West African species of the Tribe Bauhinieae (Cercidioideae)

The tribe Bauhinieae is the largest and most taxonomically complex group within the subfamily Cercidoideae. They possess the most distinguishable morphological features but are the most variable group. Here, we explore the phylogenetic relationship of the tribe Bauhinieae using morphological, anatomical and molecular data (ITS, rbcL, trnL-F, ITS+rbcL+trnL-F). Relationships inferred from morphological, anatomical and molecular data revealed congruent result, a non-monophyletic Bauhinia and Piliostigma group. The leaf epidermal shape in all Bauhinia species examined are polygonal with straight cell walls except B. tomentosa, which has an undulating cell wall. Stomatal types observed vary between the two genera studied. Bauhinia species has paracytic stomata while Piliostigma species exhibited hemiparacytic stomata. Dendrogram generated revealed the interrelationship between the species at a distance value of 80. Bayesian analysis revealed a high resolution of species and posterior probability. The strict consensus tree for all the tested gene regions revealed a polyphyletic Bauhina divided into three major clades. The Piliostigma group exhibited a paraphyletic and polyphyletic relationship within the Bauhinia group at high support values. B tomentosa exhibited a closer relationship with Piliostigma species. These results support the proposition to divide members of the large Bauhinia s.s group into subclades. This study has attempted to elucidate the unresolved species and genus level taxonomy of the tribe Bauhinieae. However, more variable gene regions in addition to broader species sampling should be considered for further phylogenetic patterns of this taxon.

Phylogenomics of the extinct Heath Hen provides support for sex-biased introgression among extant prairie grouse

Rapid divergence and subsequent reoccurring patterns of gene flow can complicate our ability to discern phylogenetic relationships among closely related species. To what degree such patterns may differ across the genome can provide an opportunity to extrapolate better how life history constraints may influence species boundaries. By exploring differences between autosomal and Z (or X) chromosomal-derived phylogenetic patterns, we can better identify factors that may limit introgression despite patterns of incomplete lineage sorting among closely related taxa. Here, using a whole-genome resequencing approach coupled with an exhaustive sampling of subspecies within the recently divergent prairie grouse complex (genus: Tympanuchus), including the extinct Heath Hen (T. cupido cupido), we show that their phylogenomic history differs depending on autosomal or Z-chromosome partitioned SNPs. Because the Heath Hen was allopatric relative to the other prairie grouse taxa, its phylogenetic signature should not be influenced by gene flow. In contrast, all the other extant prairie grouse taxa, except Attwater’s Prairie-chicken (T. c. attwateri), possess overlapping contemporary geographic distributions and have been known to hybridize. After excluding samples that were likely translocated prairie grouse from the Midwest to the eastern coastal states or their resulting hybrids with mainland Heath Hens, species tree analyses based on autosomal SNPs consistently identified a paraphyletic relationship with regard to the Heath Hen with Lesser Prairie-chicken (T. pallidicinctus) sister to Greater Prairie-chicken (T. c. pinnatus) regardless of genic or intergenic partitions. In contrast, species trees based on the Z-chromosome were consistent with Heath Hen sister to a clade that included its conspecifics, Greater and Attwater’s Prairie-chickens (T. c. attwateri). These results were further explained by historic gene flow, as shown with an excess of autosomal SNPs shared between Lesser and Greater Prairie-chickens but not with the Z-chromosome. Phylogenetic placement of Sharp-tailed Grouse (T. phasianellus), however, did not differ among analyses and was sister to a clade that included all other prairie grouse despite low levels of autosomal gene flow with Greater Prairie-chicken. These results, along with strong sexual selection (i.e., male hybrid behavioral isolation) and a lek breeding system (i.e., high variance in male mating success), are consistent with a pattern of female-biased introgression between prairie grouse taxa with overlapping geographic distributions. Additional study is warranted to explore how genomic components associated with the Z-chromosome influence the phenotype and thereby impact species limits among prairie grouse taxa despite ongoing contemporary gene flow.

Phylogenetic position of Drosophila bocainensis (Diptera, Drosophilidae) in the willistoni group and the paraphyletic status of the bocainensis subgroup

The willistoni group of Drosophila is subdivided into the alagitans, bocainensis, and willistoni subgroups, and has been an important model for studying evolutionary processes in the Neotropics for decades. Phylogenetic studies place the bocainensis subgroup as a grade in relation to the monophyletic willistoni subgroup, although these included molecular or morphological data for up to 4 species of the 12 species included in the first subgroup. Here, we characterized the first nucleotide sequences for three mitochondrial and five nuclear genes of Drosophila bocainensis Pavan & da Cunha, 1947 and employed these for addressing the paraphyly of this subgroup under a coalescent approach. Our results still recovered this paraphyletic relationship, placing D. bocainensis, Drosophila capricorni Dobzhansky & Pavan, 1943 and Drosophila sucinea Patterson & Mainland, 1944 in a basal clade, which diverged around 6.81 million years ago. The relationship of Drosophila nebulosa Sturtevant, 1916 and Drosophila fumipennis Duda, 1925 as a sister clade to the willistoni subgroup was recovered. The possible causes of such paraphyly are discussed.

PHYLOGENETIC ANALYSIS OF THE LUNGWORMS (NEMATODA: METASTRONGYLOIDEA) INFERRED USING NUCLEAR RIBOSOMAL AND MITOCHONDRIAL DNA SEQUENCES.

Phylogenetic relationships among the mammal-parasitic lungworms (Metastrongyloidea) were inferred using small- and large-subunit ribosomal DNA sequences together with 12S ribosomal mtDNA sequences. Maximum parsimony and Bayesian inference methods were used from optimal alignments and those filtered for alignment ambiguity. Analysis of 30 ingroup sequences using ribosomal DNA sequences yielded a single most parsimonious tree. Monophyly of the Metastrongyloidea was supported, but there was no support for monophyly of any of the 7 families as they have been traditionally defined. Parafilaroides decorus, an abursate lungworm of pinnipeds currently classified in the Filaroididae, was nested within a clade containing members of the Pseudaliidae, parasites of cetaceans. The tree also shows clades somewhat resembling the traditional familial divisions of the Metastrongyloidea, but in all groups, paraphyletic relationships were recovered. In a combined analysis of nuclear rDNA and 12S mtDNA, maximum parsimony and Bayesian analyses showed similar patterns to those observed with only nuclear rDNA sequences. Based on the phylogeny, the respiratory tract was inferred to be the ancestral predilection site for Metastrongyloidea, with multiple evolutionary invasions of extrapulmonary sites such as sinuses, circulatory system, and meninges. Similarly, the ancestral host was inferred to be a carnivore with subsequent colonization events into marsupial, rodent, artiodactyl, pinniped, and cetacean hosts.

Paraphyletic relationships revealed by mitochondrial DNA in the Peromyscus mexicanus species group (Rodentia: Cricetidae)

Although deer mice (Peromyscus spp.) are among the most studied small mammals, their species diversity and phylogenetic relationships remain unclear. The lack of taxonomic clarity is mainly due to a conservative morphology and because some taxa are rare, have restricted distributions, or are poorly sampled. One taxon, P. mexicanus, includes southern Mexican subspecies that have not had their systematic placement tested with genetic data. We analyzed the phylogenetic relationships and genetic structure of P. mexicanus populations using sequences of the mitochondrial gene cytochrome b. We inferred that P. mexicanus is paraphyletic, with P. m. teapensis, P. m. tehuantepecus, andP. m. totontepecus more closely related to P. gymnotis than to P. m. mexicanus. This highly divergent clade ranges from northeastern Oaxaca to northern Chiapas, including southern Veracruz, and southern Tabasco. In light of this group’s mitochondrial distinctiveness, cohesive geographic range, and previously reported molecular, biochemical, and morphological differences, we recommend it be treated as P. totontepecus. Our findings demonstrate the need for an improved understanding of the diversity and evolutionary history of these common and abundant members of North American small mammal communities.

Plastome structure of 8 Calanthe s.l. species (Orchidaceae): comparative genomics, phylogenetic analysis

BackgroundCalanthe (Epidendroideae, Orchidaceae) is a pantropical genus distributed in Asia and Africa. Its species are of great importance in terms of economic, ornamental and medicinal values. However, due to limited and confusing delimitation characters, the taxonomy of the Calanthe alliance (Calanthe, Cephalantheropsis, and Phaius) has not been sufficiently resolved. Additionally, the limited genomic information has shown incongruences in its systematics and phylogeny. In this study, we used illumina platform sequencing, performed a de novo assembly, and did a comparative analysis of 8 Calanthe group species' plastomes: 6 Calanthe and 2 Phaius species. Phylogenetic analyses were used to reconstruct the relationships of the species as well as with other species of the family Orchidaceae.ResultsThe complete plastomes of the Calanthe group species have a quadripartite structure with varied sizes ranging between 150,105bp-158,714bp, including a large single-copy region (LSC; 83,364bp- 87,450bp), a small single-copy region (SSC; 16,297bp -18,586bp), and a pair of inverted repeat regions (IRs; 25,222bp - 26,430bp). The overall GC content of these plastomes ranged between 36.6-36.9%. These plastomes encoded 131-134 differential genes, which included 85-88 protein-coding genes, 37-38 tRNA genes, and 8 rRNA genes. Comparative analysis showed no significant variations in terms of their sequences, gene content, gene order, sequence repeats and the GC content hence highly conserved. However, some genes were lost in C. delavayi (P. delavayi), including ndhC, ndhF, and ndhK genes. Compared to the coding regions, the non-coding regions had more sequence repeats hence important for species DNA barcoding. Phylogenetic analysis revealed a paraphyletic relationship in the Calanthe group, and confirmed the position of Phaius delavayi in the genus Calanthe as opposed to its previous placement in Phaius.ConclusionThis study provides a report on the complete plastomes of 6 Calanthe and 2 Phaius species and elucidates the structural characteristics of the plastomes. It also highlights the power of plastome data to resolve phylogenetic relationships and clarifies taxonomic disputes among closely related species to improve our understanding of their systematics and evolution. Furthermore, it also provides valuable genetic resources and a basis for studying evolutionary relationships and population genetics among orchid species.

Mito-phylogenetic relationship of the new subspecies of gentle monkey Cercopithecus mitis manyaraensis, Butynski & De Jong, 2020.

In 2020, a new subspecies was described in the Cercopithecus mitis complex, the Manyara monkey C. m. manyaraensis, Butynski & De Jong, 2020. The internal taxonomy of this species complex is still debated, and the phylogenetic relationships among the taxa are unclear. Here we provide the first mitochondrial sequence data for C. m. manyaraensis to determine its position within the mitochondrial phylogeny of C. mitis. This subspecies clusters within the youngest (internal divergences between 1.01 and 0.42 Ma) of three main taxonomic clades of C. mitis. Its sister lineages are C. m. boutourlinii (Ethiopia), C. m. albotorquatus (Kenya and Somalia), C. m. albogularis (Kenya and Tanzania), and C. m. monoides (Tanzania and Mozambique). In general, the phylogenetic tree of C. mitis based on mitochondrial sequence data indicates several paraphyletic relationships within the C. mitis complex. As in other African cercopithecines (e.g. Papio and Chlorocebus), these data are suitable for reconstructing historic biogeographical patterns, but they are only of limited value for delimitating taxa.

Divergence With Gene Flow and Contrasting Population Size Blur the Species Boundary in Cycas Sect. Asiorientales, as Inferred From Morphology and RAD-Seq Data.

The divergence process of incipient species is fascinating but elusive by incomplete lineage sorting or gene flow. Species delimitation is also challenging among those morphologically similar allopatric species, especially when lacking comprehensive data. Cycas sect. Asiorientales, comprised of C. taitungensis and C. revoluta in the Ryukyu Archipelago and Taiwan, diverged recently with continuous gene flow, resulting in a reciprocal paraphyletic relationship. Their previous evolutionary inferences are questioned from few genetic markers, incomplete sampling, and incomprehensive morphological comparison by a long-term taxonomic misconception. By whole range sampling, this study tests the geographic mode of speciation in the two species of Asiorientales by approximate Bayesian computation (ABC) using genome-wide single nucleotide polymorphisms (SNPs). The individual tree was reconstructed to delimit the species and track the gene-flow trajectory. With the comparison of diagnostic morphological traits and genetic data, the allopatric speciation was rejected. Alternatively, continuous but spatially heterogeneous gene flow driven by transoceanic vegetative dispersal and pollen flow with contrasting population sizes blurred their species boundary. On the basis of morphological, genetic, and evolutionary evidence, we synonymized these two Cycas species. This study highlights not only the importance of the Kuroshio Current to species evolution but also the disadvantage of using species with geographically structured genealogies as conservation units.

Morphological and molecular description of Pallisentis roparensis n. sp. (Acanthocephala: Quadrigyridae) infecting the freshwater cat fish Wallago attu from Ropar Wetland, Punjab, India.

The study describes a new species of Pallisentis Van Cleave, 1928 infecting the freshwater cat fish Wallago attu Bloch and Schneider, 1801 from Ropar wetland, Punjab, India. The morphological characters of Pallisentis roparensis include proboscis with 4 circles of 10 hooks each gradually declining in size, first circle of hooks <100 μm in length, 15–16 circles of Y-shaped collar spines and conical trunk spines present up to the posterior end in the females and the anterior region of cement gland in males. Saefftigen's pouch is present and cement gland nuclei are 22–25 in males. The sequences generated for 18S, 28S and ITS1-5.8S-ITS2 molecular markers of the newly described species are nested well among the other comparable sequences from the GenBank. The phylogenetic analyses show the monophyly of the genus Pallisentis but point towards the paraphyletic relationship among the three subgenera. The histopathology of fish intestine indicates that the parasite stimulates the inflammatory immune response causing serious injury to the mucosa and dilation of the lymphatic vessels of small intestine.

Mitochondrial DNA indicate paraphyletic relationships and resolve disjunct distributions in the Neotoma mexicana species group

Woodrats (genus Neotoma) comprise 24 species found primarily in the US and Mexico. The Neotoma mexicana species group reaches its southernmost distribution in the highlands of southern Mexico and Central America. Previous research suggested that N. mexicana has a disjunct distribution, and it, N. ferruginea and N. picta have allopatric distributions and limit each other around the lowlands of the Isthmus of Tehuantepec. However, these hypotheses were suggested with incomplete subspecific sampling near the isthmus. We used new samples of N. m. parvidens from the Oaxacan Sierra Madre del Sur and N. m. tropicalis from the Sierra Norte de Oaxaca to assess their taxonomic affinity. With new sequences of the mitochondrial cytochrome-b gene, we found that both subspecies belong to N. ferruginea, not N. mexicana. We therefore suggest that N. mexicana is continuously distributed from the US to the Transmexican Volcanic Belt, N. picta inhabits the Guerreran Sierra Madre del Sur, and N. ferruginea ranges from the Oaxacan Sierra Madre del Sur to Central America. Our findings indicate that the Isthmus of Tehuantepec did not promote speciation in these woodrats, but we did detect intraspecific genetic differentiation between samples of N. ferruginea from east and west of the isthmus.

An insight into molecular taxonomy of bufonids, microhylids, and dicroglossid frogs: First genetic records from Pakistan.

The current study was focused on documentation of amphibian assemblage in North Punjab and Islamabad Capital Territory, Pakistan, by using mitochondrial gene sequences of 16S rRNA. Our study entailed 37% of the known amphibian species of the country. We provided a phylogenetic analysis based on 74 newly generated mitochondrial 16S rRNAs from nine species of genus Microlyla, Duttaphrynus, Allopaa, Nanorana, Sphaerotheca, Minervarya, Hoplobatrachus, and Euphlyctis. We employed the maximum‐likelihood inference and Bayesian analysis to assess the taxonomic status of the samples obtained from Pakistan, with respect to other congeneric species from surrounding regions. Our findings confirmed the taxonomic status of South Asian anuran species Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Allopaa hazarensis, Nanorana vicina, Sphaerotheca maskeyi (synonym: S. pashchima), Minervarya pierrei, Hoplobatrachus tigerinus, and Euphlyctis kalasgramensis in Pakistan. We have reported new country records of genus Minervarya ( M. pierrei). Minervarya pierrei was previously misidentified as Fejervarya limnocharis, due to dearth of genetic information. We provided the first genetic records of our endemic species N. vicina. The results revealed the taxonomic placement of N. vicina with respect to its congeners and validated the taxonomic status of N. vicina from its type locality (Murree) for the first time. The findings of the present study also indicated the paraphyletic relationship of A.‐ hazarensis with Nanorana species. So, based on our phylogenetic inferences, morphological characters, and habitat preferences, validity of generic status of A. hazarensis is undecided. As our data were not enough to resolve this issue, we suggest sequencing of additional mitochondrial and nuclear genes in the future studies to get a better resolution. We recommend carrying out extensive surveys throughout the country for proper scientific documentation of amphibians of Pakistan. Many new species, some of them might be endemic to Pakistan, are expected to be discovered, and taxonomic status of other species would be resolved.

Complete mitochondrial genome of the ragworm annelid Hediste diversicolor (of Müller, 1776) (Annelida: Nereididae)

Marine annelids are a globally distributed and species-rich group, performing important ecological roles in macrobenthic communities. Yet, the availability of molecular resources to study these organisms is scarcer, comparatively with other phyla. Here, we present the first complete mitogenome of the Atlantic ragworm Hediste diversicolor (OF Muller, 1776). The mitogenome (15,904 bp long) contains 13 protein-coding genes, 22 transfer RNA, and two ribosomal RNA genes, all encoded in the same strand. Gene arrangement and composition are identical to those observed in two available congeneric species, Hediste diadroma and Hediste japonica. The phylogenetic analysis using both maximum-likelihood and Bayesian inference methods reveal a well-supported monophyly of genus Hediste and the already reported paraphyletic relationships within the subfamilies Nereidinae and Gymnonereidinae. Our results highlight the relevance of increasing the molecular sampling within this diverse group of marine fauna.

The complete mitochondrial genome of the beef cattle hookworm Bunostomum phlebotomum (Nematoda: Bunostominae)

The bovine hookworm Bunostomum phlebotomum (Nematoda: Bunostominae) is a blood-feeding nematode with important socioeconomic impact in the cattle breeding industry. In the present study, the complete mitochondrial genome sequence of a representative individual of B. phlebotomum from beef cattle in Southwest China was determined using the next generation sequencing technology. The genome was 13,799 bp in size and encoded 12 protein-coding genes, 22 tRNA genes and two rRNA genes. The phylogeny revealed that although B. phlebotomum from Chinese beef cattle and yaks were more closely related to each other than to that from Australian cows, these three bovine-originated B. phlebotomum grouped together and formed paraphyletic relationships with Bunostomum trigonocephalum (goat/sheep hookworm) and Necator americanus (human hookworm), supporting their sister-species relationships within Bunostominae. The cumulative mitochondrial DNA data provides a better understanding of phylogenetic relationships of this species in cattle.

Wide-Scale Gene Flow, Even in Insects that have Lost their Flight Ability: Presence of Dispersion Due to a Unique Parasitic Ecological Strategy of Piggybacking Hosts.

We focused on Meloe beetles that have lost all flight ability, and conducted molecular phylogeographic analyses based on their mitochondrial DNA COI and nuclear DNA EF1- α regions. Meloe beetles infiltrate bumblebee nests by attaching to bumblebees as they pollinate flowers and thereafter have a unique and specific life history as they complete their life-cycle within the host nest; flight-based dispersal is achieved by piggybacking on bumblebees. In fact, Meloe beetles, which cannot fly, even inhabit remote islands (i.e., "Oceanic Islands"). Regarding four species, i.e., Meloe coarctatus, Meloe proscarabaeus, Meloe violaceus and Meloe corvinus, the conventional morphological classification system based on morphological characteristics was strongly supported by the molecular markers. On the other hand, for two species, Meloe menoko and Meloe auriculatus, it was found that M. menoko may be evaluated as having a paraphyletic relationship with M. auriculatus. Furthermore, two other cryptic, undescribed species were also discovered in this study. One was collected in the Nikko Highland, and inhabited the area sympatrically with M. coarctatus. The other was collected from Hachijo-jima Island. These cryptic species were highly differentiated, independent lineages in terms of mitochondrial and nuclear gene regions. That is to say, a new level of species diversity was revealed among the Meloe beetle species, known for their unique and strange ecological and ethological characteristics.

Multiple data revealed two new species of the Asian horned toad Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from the eastern corner of the Himalayas.

Multiple disciplines can help to discover cryptic species and resolve taxonomic confusions. The Asian horned toad genus Megophryssensu lato as a diverse group was proposed to contain dozens of cryptic species. Based on molecular phylogenetics, morphology, osteology, and bioacoustics data, the species profiles of Megophrys toads in the eastern corner of Himalayas in Medog County, Tibet Autonomous Region, China was investigated. The results indicated that this small area harbored at least four Megophrys species, i.e., M. medogensis, M. pachyproctus, Megophrys zhouisp. nov., and Megophrys yeaesp. nov., the latter two being described in this study. Additionally, the mitochondrial DNA trees nested the low-middle-elevation and high-elevation groups of M. medogensis into a monophyletic group, being in discordance with the paraphyletic relationship between them revealed in the nuclear DNA trees. The findings highlighted the underestimated biodiversity in Himalayas, and further indicated that the Megophrys toads here have been probably experienced complicated evolutionary history, for example, introgression between clades or incomplete lineage sorting and niche divergences in microhabitats. Anyway, it is urgent for us to explore the problems because these toads are suffering from increasing threats from human activities and climatic changes.