Mitochondrial DNA Cytochrome Research Articles

Relationships between crayfish population genetic diversity, species richness, and abundance within impounded and unimpounded streams in Alabama, USA.

Understanding the relationship between multi-scale processes driving community- and population-level diversity can guide conservation efforts. While the importance of population-level genetic diversity is widely recognized, it is not always assessed for conservation planning, and positive correlations with community-level diversity are sometimes assumed, such that only the latter is measured. We surveyed species richness and cumulative multispecies abundance of crayfishes in impounded and unimpounded streams in the southern Appalachian Mountains (Alabama, USA). We simultaneously assessed levels of population genetic diversity within two focal crayfishes (Faxonius validus and F. erichsonianus) using nuclear (nDNA; inter-simple sequence repeat (ISSR)) and mitochondrial DNA (mtDNA; mitochondrial DNA cytochrome oxidase subunit I (mtCOI)) markers. We then tested for species-genetic diversity correlations (SGDCs), species diversity-abundance correlations (i.e., more individuals hypothesis, MIH), and abundance-genetic diversity correlations (AGDCs) across sites. We also examined the relationship between each of the three different types of correlation (i.e., species richness, cumulative multispecies abundance, and population genetic diversity) and stream habitat characteristics and fragmentation. Surprisingly, based on F. validus mtDNA data, sites with the greatest multispecies abundance had the lowest genetic diversity, indicating a negative AGDC. However, no AGDC was evident from nDNA. There was no evidence of SGDCs for F. validus based on either of the two genetic data types. For F. erichsonianus, there was no evidence for SGDC or AGDC. When considering the community-level data only, there was no support for the MIH. Stream width was positively correlated with F. validus genetic diversity, but negatively correlated with multispecies abundance. Similarly, species richness was positively correlated with stream width in unimpounded streams but negatively correlated with width in impounded streams. These findings indicate that community-level diversity cannot be indiscriminately used as a proxy for population-level diversity without empirically testing this correlation on the focal group. As such, community- and population-level assessments for multiple crayfish species are needed to better understand drivers of diversity and eco-evolutionary processes which will aid in the conservation of this vulnerable taxonomic group.

Genetic Diversity, Kinship, and Polychromatism in the Spotted Eagle Ray Aetobatus ocellatus of Fiji

The spotted eagle ray Aetobatus ocellatus (Kuhl, 1923) has a widespread Indo-West Pacific distribution and displays substantial population genetic structuring. Genetic data are crucial for understanding the species’ diversity, connectivity, and adaptation. However, molecular genetic information on A. ocellatus from Melanesia is lacking, which impedes our understanding of gene flow among geographic regions. In this study, we sampled 45 A. ocellatus, primarily from Fiji’s largest fish market in the capital, Suva. Mitochondrial DNA Cytochrome C Oxidase subunit I (COI) barcoding was used for species identification, and DArT-seqTM technology was applied to assess the nuclear genetic diversity. Barcoding of the COI gene showed a 98.6% to 99.8% similarity to A. ocellatus reference sequences in the Barcode of Life Data System, and the 45 individuals were represented by three major evolutionary haplotype clusters. Genotyping resulted in 24,313 Single-Nucleotide Polymorphisms (SNPs) which were quality-filtered to 7094 SNPs per individual. The observed heterozygosity level was 0.310. The inbreeding coefficient was positive, and genotyping identified one full-sibling pair and one half-sibling pair from the 45 individuals. Additionally, eagle rays exhibit polychromatic patterns, and at least three ventral pattern variations were recorded in specimens from the market. Collectively, our main findings characterize the genetic profile of A. ocellatus in Fiji and can help to understand the diversification of this species within the region.

Genetic Diversity and Distribution of Haplotypes of Freshwater Eel in Baltic Lakeland Based on Mitochondrial DNA D-Loop and Cytochrome b Sequence Variation

The European eel (Anguilla anguilla (L.)) is a unique catadromous euryhaline fish and the only anguillid eel species classified as ‘critically endangered’. The Baltic Lakeland area contains naturally recruited and introduced eels. There is great uncertainty regarding a baseline for the location and number of naturally dispersed eels in the region. Little is known about the genetic structure of the European eel populations in Baltic countries. The estimation of population genetic structure is important for the efficient management of naturally recruited and introduced eels. Two mitochondrial regions were used to investigate the genetic structure within and between eel samples from 11 waterbodies. In this study, new, unique, and widely distributed haplotypes were revealed. The studied eel population in the Baltic Lakeland shows high genetic diversity, which is possibly a result of intensive restocking programs. Sequences characterized for Anguilla rostrata were revealed in both mitochondrial regions. Understanding the genetic structure of eel populations worldwide is crucial for conservation efforts. Eel restocking in waterbodies where natural migration is restricted contributes to diversity loss for the world gene pool of eels.

DNA barcoding confirms the presence of invasive Parachanna obscura and rare species in the Mweru-Luapula fishery

DNA barcoding has recently been instrumental in identifying both invasive and undetected species in aquatic environments. This study aimed at analysing collected fish fin clips to ascertain the identity of native species and the Parachanna species that have invaded the Mweru-Luapula (ML) fishery of Zambia. The identification process was carried out through field phenotypic analysis using species guides and DNA barcoding, with the mitochondrial DNA (mtDNA) cytochrome C oxidase 1 (COI) gene fragment. Of the 28 specimens for which DNA was successfully PCR amplified, five matched the reference sequences of species and 22 matched the reference sequences of genera on the NCBI GenBank. Five rare species, namely Oreochromis niloticus, Coptodon zillii, Mormyrus kannume, Thoracochromis buysi and Tylochromis polylepis were identified. The study further affirmed the presence of invasive Parachanna obscura in the fishery and its interconnected water bodies. Parachanna obscura invaded the fishery through annual flooding from aquacultural facilities in the Democratic Republic of Congo (DRC). There is a need to investigate this invasion further by using large specimen sample sizes and also by applying the gonadosomatic index (GSI) to determine invasive species occupancy and impact on native species throughout the fishery. This study provides a platform for further detailed taxonomic verification and species inventory of the entire ML fishery. This will facilitate the development of a viable and sustainable strategy to appropriately curb the impact of invasive species, and will thus contribute to the conservation of the ML aquatic biodiversity.

Bionomic characterization of Anopheles mosquitoes in the Ethiopian highlands and lowlands

BackgroundThe protective effectiveness of vector control in malaria relies on how the implemented tools overlap with mosquito species-specific compositions and bionomic traits. In Ethiopia, targeted entomological data enabling strategic decision-making are lacking around high-risk migrant worker camps in the lowlands and resident communities in the highlands—resulting in suboptimal malaria control strategies for both populations. This study investigates spatial and temporal mosquito behavior, generating baseline evidence that will improve malaria control for both migrant workers in the lowlands and their home communities in the highlands.MethodsHourly Centers for Disease Control and Prevention (CDC) light trap collections were performed indoors and outdoors during the peak (October to December 2022) and minor (March to May 2023) malaria transmission seasons. These seasons coincide with the post-long rain and post-short rain seasons, respectively. Eight resident households were sampled from each of four villages in the highlands and eight households/farm structures on and near farms in four villages in the lowlands. The sampling occurred between 18:00 and 06:00. Spatiotemporal vector behaviors and hourly indoor and outdoor mosquito capture rates, used as a proxy for human biting rates, were calculated for overall catches and for individual species. Adult mosquitoes were identified using morphological keys, and a subset of samples were confirmed to species by sequencing ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or mitochondrial DNA cytochrome c oxidase subunit 1 (Cox1).ResultsIn the highlands, 4697 Anopheles mosquitoes belonging to 13 morphologically identified species were collected. The predominant species of Anopheles identified in the highlands was An. gambiae sensu lato (s.l.) (n = 1970, 41.9%), followed by An. demeilloni (n = 1133, 24.1%) and An. cinereus (n = 520, 11.0%). In the lowland villages, 3220 mosquitoes belonging to 18 morphological species were collected. Anopheles gambiae s.l. (n = 1190, 36.9%), An. pretoriensis (n = 899, 27.9%), and An. demeilloni (n = 564, 17.5%) were the predominant species. A total of 20 species were identified molecularly, of which three could not be identified to species through comparison with published sequences. In highland villages, the indoor Anopheles mosquito capture rate was much greater than the outdoor rate. This trend reversed in the lowlands, where the rate of outdoor captures was greater than the indoor rate. In both highlands and lowlands, Anopheles mosquitoes showed early biting activities in the evening, which peaked between 18:00 and 21:00, for both indoor and outdoor locations.ConclusionsThe high diversity of Anopheles vectors and their variable behaviors result in a dynamic and resilient transmission system impacting both exposure to infectious bites and intervention effectiveness. This creates gaps in protection allowing malaria transmission to persist. To achieve optimal control, one-size-fits-all strategies must be abandoned, and interventions should be tailored to the diverse spatiotemporal behaviors of different mosquito populations.Graphical

Non‐destructive molecular methods to identify Monochamus alternatus (Coleoptera: Cerambycidae), a major vector of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

AbstractMonochamus alternatus is one of the most important borers of conifers and the main vector of the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease. It causes massive death of pine trees and seriously affects the health of forest ecosystems. Traditionally, adults or larvae are identified by morphological or molecular methods. However, diagnosing M. alternatus larvae at different instars collected from forests is expensive and time‐consuming. Non‐destructive molecular diagnostic protocols are, therefore, being developed to detect biological traces (i.e. exuviae, excreta) and to determine the distribution and spread of this pest. In this study, based on the alignment of mitochondrial DNA cytochrome oxidase subunit I (COI) gene sequences of M. alternatus and other insect species, we designed the primer pair of Mal‐SF/Mal‐SR and probe of Mal‐P for M. alternatus. TaqMan probe‐based qPCR was developed to identify the occurrence of M. alternatus in forests by amplifying the DNA samples obtained from its adult, larva, frass, excreta and exuviae. The amplification results were very effective. The lowest amount of M. alternatus DNA that could be detected with a Cq of 31.93 in the mixed samples was 0.64 pg, showing very high sensitivity. This assay can easily identify M. alternatus from other non‐target wood‐borer species using its frass and exuviae, providing a new diagnostic protocol for monitoring the occurrence and distribution of M. alternatus in forests.

Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing.

Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.

Mitochondrial DNA (mtDNA) genetic variability of cytochrome B gene (CYTB) in three populations of native rabbits in Egypt

The current study aimed to detect the genetic variability of mitochondrial DNA (mtDNA) cytochrome B (CYTB) gene in 80 native Egyptian rabbits (NER) belonging to three populations in three different agriculture regions (Delta, Middle, and Upper Egypt) and also investigate the origin of these breeds by phylogenetic relationship analysis. A total of 62 haplotypes were recorded among the three NER populations. The native Upper Egypt rabbits (NUER) expressed the highest number of haplotypes, mutations, polymorphic sites, and haplotype diversity (23, 55, 51, and 1.00, respectively). While it gave the lowest values of nucleotide diversity (0.0262) and Tajima's D (−0.01435), the percentage of mutual haplotypes was 4.8 % between the Middle and Delta Egypt populations. The phylogenetic analysis showed that there were more separated haplotypes of NUER compared to other populations. The same finding was also observed when supported by 31 sequences of different rabbit breeds retrieved from the GeneBank database. A mutual haplotype was observed between native Delta Egyptian rabbits (NDER) and New Zealand (NWZ) rabbits. The results of this study shed light on the importance of indigenous breeds in rural areas through mtDNA, which contributes to finding sustainable strategies to conserve and improve genetic resources in Egyptian rural areas.

DNA barcoding of common freshwater eleotrids (Gobiiformes: Eleotridae) in the Philippines, with a global comparison and notes on their conservation and management

Eleotrids, also known as gudgeons or sleeper gobies, are found mostly in freshwater and mangrove habitats of tropical and subtropical regions. Some eleotrids exhibit minimal morphological variation at the species level. In this study, DNA barcoding was used to assess the molecular diversity of common freshwater eleotrids in the Philippines in relation to other eleotrid species from other countries with available DNA sequences in public databases. A total of 422 mitochondrial DNA cytochrome c oxidase I (COI) gene sequences were analyzed: 107 of which were generated from this study, while the remaining 315 sequences were mined from Barcode of Life Data System and GenBank. Both the neighbor-joining and maximum likelihood trees constructed based on the COI sequences clustered most of the species in concordance with their taxonomic classification. The mean Kimura 2-parameter genetic distances within species and genera were 1.31% and 11.60%, respectively. As expected, the mean interspecific distance was higher than the mean intraspecific distance except in some species. This study revealed the presence of nine distinct eleotrid species belonging to five genera in the Philippines. These species include Giuris laglaizei, G. tolsoni, Eleotris acanthopomus, E. fusca, E. melanosoma, Belobranchus sp., Belobranchus belobranchus, Butis butis, and Ophiocara ophicephalus. This study also revealed the presence of possibly undescribed Belobranchus species in the Philippines. This undescribed Belobranchus species differs by a high percentage of genetic divergence from the other species of Belobranchus (3.00% from B. segura and 13.40% from B. belobranchus). Furthermore, high genetic distance (2.62%) was also observed between G. laglaizei species of Laguna de Bay and Mindanao. This study strongly recommends a taxonomic review of the Laguna de Bay G. laglaizei and the Philippine Belobranchus spp., as these species may harbor species complexes that require closer examination so that proper conservation and management measures can be implemented for each species.

Historical imprints on genetic population structure in direct-developing beach whelks (Bullia digitalis)

Marine organisms often show deep genetic divergence between lineages that are characterized by shallow population structure. The distinction of historical and contemporary demographic forces in creating and maintaining these genetic patterns is crucial for management and conservation. In this study, we examined the genetic population structure of Bullia digitalis, a lecithotrophic sandy beach whelk that lacks a pelagic larval phase and is thus expected to show limited connectivity between populations. Mitochondrial (mt)DNA cytochrome c oxidase subunit I (COI; 540 bp, n = 214) and a reanalysis of allozyme data (14 loci, n = 735) among populations along the cool southeast Atlantic (west) and warm-temperate southwest Indian Ocean (south) coasts of southern Africa showed a strong phylogeographic break across Cape Point with mtDNA (ΦRT = 0.6, p < 0.001), but a weak break with allozymes (FRT = 0.002, p = 0.004). MtDNA gene flow occurred asymmetrically from the west to the south coast. Although there was no mtDNA differentiation between populations on either side of that break (ΦSR = 0.0), there was weak but significant isolation by distance (IBD) among south coast samples (r2 = 0.20, p = 0.039). In contrast, significant allozyme differentiation was detected on small spatial scales but without overall IBD. MtDNA mismatch analysis indicated a recent range expansion, suggesting historic population expansions, most likely during periods of lower sea levels and greater habitat continuity along southern African shores. Thus, the apparent mtDNA connectivity among populations of beach whelks likely reflects historic, rather than contemporary, gene flow.

Genetic variability of Aedes aegypti (Diptera: Culicidae) in El Salvador and Honduras: presence of a widespread haplotype and implications for mosquito control

BackgroundThis study examined population genetics of Aedes aegypti in El Salvador and Honduras, two adjacent countries in Central America. Aedes aegypti is associated with yellow fever, dengue, chikungunya, and Zika. Each year, thousands of cases of dengue are typically reported in El Salvador and Honduras.MethodsIn El Salvador, collections were obtained from five Departments. In Honduras, samples were obtained from six municipalities in four Departments. Mitochondrial DNA cytochrome oxidase I (COI) was sequenced, and consensus sequences were combined with available sequences from El Salvador to determine haplotype number, haplotype diversity, nucleotide diversity, and Tajima’s D. A haplotype network was produced to examine the relationship between genotypes.ResultsIn El Salvador, there were 17 haplotypes, while in Honduras there were 4 haplotypes. In both El Salvador and Honduras, Haplotype 1 is most abundant and widespread. In El Salvador, haplotype H2 was also widespread in 10 of 11 sampled municipalities, but it was not present in Honduras. The capital of El Salvador (San Salvador) and the eastern region of ES had the highest haplotype diversity of regions sampled.ConclusionsHaplotype 1 and H2 each belong to different phylogenetic lineages of Ae. aegypti. The most geographically widespread haplotype (H1) may have been present the longest and could be a remnant from previous eradication programs. These data may contribute to future control programs for Ae. aegypti in the two countries.Graphical

Hyperoxia impacts heme oxygenase 1 and mitochondrial quality control in a rodent model of sarcopenic obesity

Critically ill sarcopenic obese (SO) patients may be at higher risk for other complications and require mechanical ventilation, extending length of stay and recovery time. It may be because in sarcopenia and obesity, oxidants, inflammation and mitochondrial fragmentation increase, impacting respiration. Heme oxygenase 1 (HO-1) is an enzyme critical for cellular antioxidant and anti-inflammatory defenses, and in the regulation of mitochondrial quality control (MQC). Whether HO-1 signaling is impaired in SO during heightened oxidant stress remains unknown, the hypothesis of this research. Young (7-week-old) and old (72-week-old) C57BL/6J mice consumed high-fat (58%) or control (11% fat) diets for 16-weeks. Mean body weight (g), glucose AUC and strength (N) (±SD) measured after dietary interventions were as follows: young lean (33±2, 540±220, 1.9±0.3), young obese (53±3, 710±116, 1.8±0.4), old lean (38±4, 597±148, 1.5±0.3), and old obese (57±10, 654±119, 1.4±0.2). To induce oxidant stress, mice were exposed to 100% oxygen for 48 h. Lower hindlimb skeletal muscle was harvested 16 h later. Oxidant stress and inflammation were determined by measuring superoxide (DHE), DNA oxidation (8-OHdG), mitochondrial DNA lesions and cytochrome b mRNA, and IL-10, IL-1β and TNF-α protein expression. MQC was assessed by measuring regulators of mitochondrial fission (DRP1) and fusion (MFN1 and 2), mitophagy (PINK1, Parkin and LC3-II), and mitochondrial biogenesis (NFE2L2, PGC-1α, NRF1 and 2, and TFAM). The mitochondrial membrane potential was measured using TMRM. In contrast to air controls where HO-1 protein expression was increased by aging and obesity, hyperoxia caused substantial reductions in both HO-1 protein and mRNA within old and obese mice. The greatest reductions were found in SO (25% of young lean). The changes in HO-1 were accompanied by the following responses to hyperoxia: One, superoxide and DNA oxidation increased in old and obese mice by as much as 200%, leading to elevated mitochondrial DNA lesions and reduced cytochrome b. Two, IL-1β and TNF-α increased in aged mice by 41-129% over young mice, whereas IL-10 was lower (-33%) only in SO. Three, regulators of mitochondrial fission and fusion and mitophagy were reduced by aging and obesity, being lower in sarcopenia which may explain why mitochondrial LC3-II was only reduced in these mice. Fourth, modulators of mitochondrial biogenesis were decreased by aging and obesity, suggesting a reduced mitochondrial population. Lastly, the increased mitochondrial membrane potential measured in young obese (78%) and old lean (58%) mice was not observed in SO, indicating reduced mitochondrial activity. In sarcopenic and obese mice exposed to hyperoxia, oxidant stress and mitochondrial DNA fragmentation increase without a concomitant change in MQC to maintain a healthy and functional mitochondrial network. Suppression of HO-1 may be a mechanism for these changes during periods of hyperoxic stress in sarcopenia and obesity that contribute to poor outcomes in critical illness. This work was supported by the Developing Research Excellence in Anesthesia Management Innovation Grant (2021). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ngāokeoke Aotearoa: The Peripatoides Onychophora of New Zealand.

(1) Background: Originally described as a single taxon, Peripatoides novaezealandiae (Hutton, 1876) are distributed across both main islands of New Zealand; the existence of multiple distinct lineages of live-bearing Onychophora across this spatial range has gradually emerged. Morphological conservatism obscured the true endemic diversity, and the inclusion of molecular tools has been instrumental in revealing these cryptic taxa. (2) Methods: Here, we review the diversity of the ovoviviparous Onychophora of New Zealand through a re-analysis of allozyme genotype data, mitochondrial DNA cytochrome oxidase subunit I sequences, geographic information and morphology. (3) Results: New analysis of the multilocus biallelic nuclear data using methods that do not require a priori assumptions of population assignment support at least six lineages of ovoviviparous Peripatoides in northern New Zealand, and mtDNA sequence variation is consistent with these divisions. Expansion of mitochondrial DNA sequence data, including representation of all existing taxa and additional populations extends our knowledge of the scale of sympatry among taxa and shows that three other lineages from southern South Island can be added to the Peripatoides list, and names are proposed here. In total, 10 species of Peripatoides can be recognised with current data.

Genetic diversity and population structure of Kawakawa Euthynnus affinis (Cantor, 1849) in Malaysia and its surrounding waters inferred by mitochondrial DNA cytochrome oxidase I and cytochrome b genes

Kawakawa (Euthynnus affinis) is a widely distributed epipelagic tuna, considered among the most economically significant species in the subtropical and tropical waters of the Indo-Pacific region. However, the lack of comprehensive information on the stock structure, management, and conservation of E. affinis populations in Malaysia raises concerns of potential overfishing and depletion of this marine fish. To ensure effective and successful species management, it is imperative to conduct a molecular- based assessment of the stock structure. In the present study, the genetic diversity and population structure of E. affinis in Malaysia and its surrounding waters were investigated using mitochondrial DNA cytochrome oxidase I (COI) and cytochrome b (Cyt b) genes. A total of 390 (COI) and 414 (Cyt b) individuals of E. affinis were sampled from 20 distinct locations situated within five geographic regions: the Straits of Malacca, South China Sea, Sulu Sea, Celebes Sea, and the Arabian Sea. The results revealed that the genetic divergence among 20 populations of E. affinis varied from low to high. The average haplotype diversity (Hd) and nucleotide diversity (π) for COI were calculated to be 0.5131 and 0.0043, respectively. On the other hand, the average haplotype diversity (Hd) and nucleotide diversity (π) for Cyt b were found to be 0.7450 and 0.0162, respectively. The analysis of haplotype distribution revealed that a total of 20 distinct haplotypes were identified for both the COI and Cyt b genes across 20 populations of E. affinis. The analysis of the Neighbor-Joining (NJ) tree and the Minimum Spanning Network (MSN) revealed the formation of three distinct clades among thesamples. AMOVA analysisshowed a significant genetic structure among the 20 populations of E. affinis, as indicated by the COI: FST = 0.5364 (P < 0.05) and Cyt b: FST = 0.9521 (P < 0.05). The neutrality test and mismatch distribution analysis indicated that the E. affinis specimens underwent a period of population expansion. This study marks a significant milestone as the first comprehensive documentation of genetic diversity and population structure of E. affinis across various locations in Malaysia, with an additional pioneering investigation conducted in the waters of Yemen.

Slow and steady saves the race: molecular and morphological analysis of three new cryptic species of Iberus land snails from the Iberian Peninsula

The Iberian Peninsula constitutes a diversity hotspot with a high number of endemisms, where the land snail genus Iberus is likely the best example. Despite this, its species diversity is still debated as it holds several cryptic species. In the present paper, we use molecular evidence (mitochondrial DNA cytochrome oxidase subunit I) to clarify the position of I. ortizi and three new cryptic species that are described herein: I. giennensis, I. axarciensis and I. antikarianus spp. nov. For this, we sampled 281 sampling points to delimitate a comprehensive geographic mapping of these species. Moreover, we carried out a comprehensive morphometric analysis based on 3205 shells. Our findings show that, morphologically, the three described species overlap in the form of their shells, their morphologies being very similar to other close species with nearby distributions (I. ortizi, I. angustatus and I. marmoratus loxanus). Still, all these species are well-defined by genetic distances, but display allopatric distributions, suggesting that they evolved by allopatric speciation as a consequence of biogeographic isolation. Hence, our findings show insights into the evolution of land snails in southeastern Spain, with implications for their conservation, given that our exhaustive sampling shows that the three species described here have very limited distribution ranges, especially I. antikarianus sp. nov. Our study, moreover, implies an integrated approach to the study of the evolution of land snails, including the sampling of the complete geographic area occupied by the genus, genetic analysis to delimit the actual species range, as well as morphometric analyses to understand the phenotypic differentiation and adaptations of the three new species.

Phylogeographic History of Endangered Hokuriku Salamander, Hynobius takedai (Amphibia: Caudata).

Knowledge of the phylogeographic history of organisms is valuable for understanding their evolutionary processes. To the best of our knowledge, the phylogeographic structure of Hokuriku salamander, Hynobius takedai, an endangered species, remains unclear. This study aimed to elucidate the phylogeographic history of H. takedai, which is expected to be strongly influenced by paleogeographic events. Phylogenetic analysis based on partial sequences of the mitochondrial DNA cytochrome b gene confirmed the genetic independence of H. takedai, and the divergence time with closely related species was estimated to be from the Late Pliocene to the Early Pleistocene. In the phylogenetic tree, two clades were identified within H. takedai, and their haplotypes were found in samples collected from the west and east of the distribution range. These intraspecific divergences were strongly influenced by geohistorical subdivisions of the current major distribution areas in the Middle Pleistocene. One clade was further subdivided and its formation may have been influenced by sea level changes in the Late Pleistocene.

Deep mitochondrial phylogeographical pattern: cryptic population structure within an ecological niche in the near-threatened Himalayan vulture (Gyps himalayensis)

Abstract The Himalayan vulture (Gyps himalayensis) has higher environmental specificity than other species in the same genus, and its population has declined due to diclofenac exposure and shortage of carcasses as a food source. Previous analysis of mitochondrial (mt) DNA cytochrome b (Cyt b) sequences reported no distinct geographical population structure in the Himalayan vulture. Contradictory results were obtained in the present study, which investigated the population structure of 14 Himalayan vultures that migrated to Thailand during winter. Using samples obtained between 2010 and 2021, as well as limited GenBank samples, the study found that the Cyt b locus was incapable of distinguishing population structure. This dataset contrasted with the mtDNA control region (CR) and Cyt b + CR dataset, which divided them into two groups, as explained by the Middle–Late Pleistocene climate change scenario. The species split into populations from the central, western, and northern regions of its distribution range within the highland vulture ecological niche. This study reviews the different methods used as compared with previous proposals for defining guidelines for the conservation of this near-threatened scavenging species.

Complex geohistory of continental islands advanced allopatric evolution even for the highly dispersive generalist red fox (Vulpes vulpes): multiple phylogenetic groups in the Japanese Archipelago

Abstract The red fox (Vulpes vulpes) plays a key role as an apex-generalist predator in terrestrial ecosystems. We estimated the phylogeographic structure, time to most recent common ancestor (tMRCA), and demographic dynamics based on the mitochondrial DNA cytochrome b gene and partial D-loop region sequences of 182 red foxes in the Japanese Archipelago, and discussed the geohistory and biotic interactions that influenced them. The Hondo red fox (Vulpes vulpes japonica), distributed on Honshu, Shikoku, and Kyushu islands, was supported as a monophyletic group. The tMRCA of the Hondo clade was ~0.148 (95% highest posterior density: 0.236–0.080) Ma. The Hondo clade diverged into two subclades, and each was roughly distributed on the eastern or western area of the Japanese Archipelago. The effective population size of the Hondo red fox remained nearly constant until ~0.03–0.02 Ma; thereafter, it grew ~10-fold. The Kita red fox (Vulpes vulpes schrencki) distributed on Hokkaido Island formed a polyphyletic group, not including the Hondo clade. The completely different phylogenetic structures of the Hondo and Kita red fox indicate that they have independent evolutionary backgrounds. These findings provide crucial insights into the formation mechanisms of diversity and endemism of mammals on continental islands.

Distribution and genetic diversity of the invasive pest Halyomorpha halys (Hemiptera, Pentatomidae) in Belgium

The brown marmorated stink bug, Halyomorpha halys, native to East Asia, is an invasive pest of economic importance. It has invaded North America and many European countries and is further expanding its range. In Belgium, it was first observed in 2011. Halyomorpha halys is known to cause severe damage in commercial fruit orchards and vegetable crops. A dramatic and unmitigated expansion of H. halys in its adventive range could lead to significant economic implications for agricultural production. In this study, occurrence data of H. halys since its first observation in Belgium was analysed together with molecular information to map the populations and evaluate the genetic diversity of this pest. The genetic diversity of H. halys in Belgium was compared to data from other invaded and native countries reported in previous studies to identify possible invasion routes. The analysis of 1176-bp of mitochondrial DNA cytochrome c oxidase I and II genes (COI and COII) led to the discovery of two novel COI-COII haplotype combinations currently unique to Belgium. The invasion of H. halys in Belgium is likely the result of multiple and ongoing introductions from its native region and from already invaded countries in Europe, particularly Italy. The expansion of the brown marmorated stink bug populations in Belgium is recent and ongoing. Presently, it appears to thrive best in northern Belgium.

Morphological differences yet genetic similarity among seasonal cohorts of Japanese anchovy during the early life stages in the Kii Channel

Abstract Species in the genus Engraulis show extensive intraspecific as well as interspecific morphological and genetic diversity. Since morphological differences do not necessarily correspond to genetic differences, it is necessary to clarify the relationship between morphological differences and genetic differences for a better understanding of the population structure. Fish morphology at a given standard length differs between cohorts of Japanese anchovy Engraulis japonicus during the early life stages in the Kii Channel, but it is unknown whether the differences are caused by genetic differences or not. The Kii Channel includes the boundary between the Pacific (southern side of the Kii Channel) and the Seto Inland Sea stocks (northern side), but stock separation is based primarily on demographic characteristics. In the present study, genetic analyses were conducted to examine the genetic differences among samples (month and area) based on mitochondrial DNA cytochrome b region (Cyt b), control region (CR) and microsatellite markers. AMOVA revealed that the percentage of genetic variation among samples was low at 0.11% for Cyt b, 0.30% for CR and 0.00% for microsatellite, and no significant genetic variation was observed among samples. Although two clades were identified in the unrooted neighbour-joining tree for Cyt b and CR, both Cyt b and CR sequences were similar between months and between areas. Accordingly, the morphological differences among cohorts can be attributed to phenotypic plasticity. Additionally, there were no genetic differences between samples from the southern side and the northern side of the Kii Channel, suggesting strong genetic connectivity in these areas.