Barcoding old Korean lepidopteran specimens using newly designed specific primer pairs

Currently, DNA barcodes are often required to be analyzed using old museum specimens when they are the only available specimens for rare or endangered species, or even type series. In this study, using eight universal primers and newly designed 315 species-specific primers, we tried to recover full-length barcode sequences from 45 dried specimens of 36 butterfly species collected between 1959 and 1980 in Korea. The eight universal primers failed entirely in the PCR amplification and sequencing of all the specimens. On the other hand, 284 primer pairs consisting of the 315 primers, targeting fragments of 71–417 bp, amplified various lengths of barcode sequences from all specimens. The fragments were successfully combined to generate the barcode sequences ranging from 444 bp to 658 bp. Notably, of the 284 primer pairs, 26 primer pairs designed for Limenitis camilla, Argynnis niobe, and Brenthis daphne successfully amplified the barcode sequences of congeneric species, Limenitis doerriesi, Argynnis nerippe, and Brenthis ino, suggesting that the species-specific primers can be available for analyzing barcode sequences of closely related species. Our study reveals that the newly designed species-specific primers will be effective in acquiring COI sequences from old butterfly specimens.     

Evaluating the DNA barcodes for identification of butterflies from Western Himalayas,Uttarakhand, India

DNA barcodes in species tagging have become a popular tool for taking inventories of species from different groups worldwide. The present study aimed to generate DNA barcodes of butterfly species from the Western Himalayas in Uttarakhand, India. The Indian Western Himalayan region (IWHR) has been explored to a limited extent about butterfly species’ diversity. However, the IWHR is prone to environmental change, and slight variations in climatic conditions can influence species diversity and change butterflies' range. The mitochondrial cytochrome c oxidase I (COI) gene was first used to generate the DNA barcode for butterflies from this region on a broad scale. 28 morphologically identified species, consisting of 102 sequences, were finally grouped into 26 species, with only two species showing ambiguity in species identification. These species had < 3% sequence variations from their neighboring relatives, suggesting cryptic species diversity. Generated sequences were also compared with the GenBank data of conspecific geographical locations, which showed intraspecies variation ranging from 1.3% to 7.3%. It was also noted that butterfly species have both intra and interspecies sequence divergence. In the phylogenetic-based species identification, a total of 28 species belonging to 4 families of butterflies were successfully discriminated, and two species at the genus level, which had high intra-specific divergence (0.025), were considered. However, the high intra-species sequence divergence observed may represent the presence of hidden species.     

Developing a new molecular marker for aphid species identification: Evaluation of eleven candidate genes with species-level sampling

The mitochondrial cytochrome c oxidase subunit I (COI) gene has been utilized as a molecular marker for aphid species identification. However, this gene has sometimes resulted in misidentification because of low interspecific genetic divergences between some species pairs. In this study, to propose new molecular markers for the family Aphididae, we first screened 2289 sequences of 11 genes (COI, COII, CytB, ATP6, lrRNA, srRNA, ITS1, ITS2, EF1a, 18S, and 28S) collected from the GenBank. Among the 11 genes, ATP6 gene revealed the largest genetic divergence among congeneric species with the smallest divergence among conspecific individuals; in contrast, species pairs with low genetic divergences (< 1%) were not observed. Secondly, for statistically testing the usefulness of ATP6 gene in species identification, we analyzed genetic distances between all of the combinations of 32 individuals of 20 species for both COI and ATP6 genes. The ATP6 gene showed lower intraspecific (on average 0.08%) and higher interspecific (on average 8.28%) genetic distances than the COI gene (on average 0.19% and 6.24%, respectively) for the same pairs of individuals. This study corroborates the usefulness of the ATP6 gene as a new molecular marker that could improve the misidentification problems that are inherent with the COI gene.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

Mitochondrial evidence uncovers a refugium for the fat dormouse (Glis glis Linnaeus, 1766) in Hyrcanian forests of northern Iran

Fat dormouse is a squirrel-like rodent which is closely tied to deciduous forest ecosystem in southwestern Eurasia. As such it is a valuable indicator of forest survival in refugia during glacial-interglacial periods. Previous phylogeographic analyses uncovered divergent fat dormouse lineages in southern refugia in Italy and the Balkans, but retrieved a surprisingly low overall genetic diversity across the majority of the species’ range. We explored 812 bp long fragment of a cytochrome b (cyt b) gene in ten fat dormice from refugial Hyrcanian forests in northern Iran. We identified 10 new cyt b haplotypes, which generated a total dataset of 28 fat dormouse haplotypes. The phylogenetic reconstruction clustered the new haplotypes into the Iranian lineage which hold a sister position against all other fat dormouse haplotypes from Europe and Asia Minor. The divergence between these lineages suggests a fragmentation event of an ancestral population at 5.76 mya (95% HPD = 3.21–8.92). This early evolutionary divergence was possibly triggered in the Middle East by dramatically divergent environmental conditions at the Messinian Salinity Crisis. The divergence clearly exceeds the intraspecific divergence, and is well within the range between congeneric rodent species. We suggest a long-term persistence of the Iranian lineage in the Hyrcanian refugium which is consitent with a high number of endemics along the southern Caspian coastal areas.     

Mitochondrial DNA sequence variation of the bumblebee,Bombus ardens (Hymenoptera: Apidae)

The bumblebee, Bombus ardens, is a valuable natural resource, and is most notably utilized for greenhouse pollination. In order to gain a greater understanding of the population genetic structure and the genetic diversity of this species, we sequenced a portion of the mitochondrial COI gene corresponding to the “DNA Barcode” region (658 bp) from 160 individuals collected over 15 Korean localities. Uncorrected pairwise distances among the eight haplotypes suggested low intraspecific genetic diversity, with a maximum sequence divergence of 0.3%. Such a low level of intraspecific genetic diversity was further reflected in local populations, particularly to islet populations, such as those of Youngheungdo, Jakyakdo, and Ulleugdo, which had zero genetic diversity. Geographically, one haplotype (BARBA01) was widespread and dominant, with a frequency of 90.6% (145 among 160 individuals). Other haplotypes were restricted to one to three localities and had low frequency. Overall, a very high rate of per generation female migration ratio (Nm = 4.6 to infinite) and a very low level of genetic fixation (F_ST = 0 to 0.099) were detected between pairs of localities, suggesting that the B. ardens populations on the Korean peninsula are panmictic, which is consistent with our understanding of their dispersal capability.     

Species diversity of butterflies in Changbai Mountain in China

Using a modified belt transect method, we investigated the butterfly communities in five different vertical vegetation belts of Changbai Mountain in China from 1992 to 2009; these belts were broadleaf deciduous forest, coniferous–deciduous mixed forest, coniferous forest, erman’s birch forest and alp tundra. We determined the number of species and abundance of butterflies in each belt and in the coniferous–deciduous mixed forest belt, we also compared these parameters among different months. Preston’s lognormal distribution was used to model the species abundance distributions and five indicators (Shannon–Wiener diversity index (H′), Pielou uniformity index (J), Simpson predominance centralization index (C), Margalef abundance index (E) and Jaccard similarity coefficients) were used to analyze the butterfly community diversity. We found four main results. (1) Across all five vertical vegetation belts, 9641 butterflies were collected, belonging to 7 families, 98 genera and 196 species. As altitude increased, the number of butterfly genera and species gradually reduced. There was a relationship between the distribution of dominant species and the total species between each belt and the distribution of vascular plants. (2) The species abundance distribution was successfully modeled as a Preston’s lognormal distribution; the best fit was obtained when α = 0.326, the determinant coefficient of the equation was 0.74798. The species abundance distribution indicates that Changbai Mountain provides a suitable environment for butterflies; there was high species richness and an even distribution of butterfly species. There were few very common and very rare species, with most species having an intermediate abundance. (3) As altitude increased, H′ and E gradually became smaller, while C showed the opposite pattern, and J did not significantly change. The similarity coefficients analysis demonstrated a clear difference among belts; the farther apart any two belts, the smaller the similarity coefficient, indicating less similarity in the butterfly communities. The similarity coefficient between the deciduous forest and the coniferous–deciduous mixed forest belt was the largest (0.651) while that between the deciduous forest and the alp tundra was the smallest (0.141). (4) Comparison of the butterfly species communities among different months in the coniferous–deciduous mixed forest found that H′ and E showed similar directional changes, while the opposite pattern was found with C; the changes in J did not necessarily reflect the actual change in diversity.     

Structural indicators of richness and rarity of epiphytic lichens on Corylus avellana in two different forest types within a nature reserve in south-western Norway

Abstract:Epiphytic lichens (and some non-lichenized fungi) on 34 coppices (204 stems) ofCorylus avellana were investigated in a 140 ha study area in south-western Norway. A total of 65 species were recorded on a total bark area of 63 m². Corylus in broad-leaved deciduous forest supported more species of macrolichens, and fewer species of microlichens, than Corylus in pine forest. The macrolichen flora of the deciduous forest differed from that of the pine forest by having a rich flora of species belonging to the Lobarion alliance. OldCorylus coppices with tall stems (>8 m), large girth (>8 cm diameter at breast height) and a noticeable cover of macrolichens (>10% of bark area) supported the highest number of rare species, and overall, species of macrolichens. More than 50% cover of microlichens indicated richness and rarity of microlichens on Corylus.     

Impacts of forest gaps on butterfly diversity in a Bornean peat-swamp forest

Forest degradation is leading to widespread negative impacts on biodiversity in South-east Asia. Tropical peat-swamp forests are one South-east Asian habitat in which insect communities, and the impacts of forest degradation on them, are poorly understood. To address this information deficit, we investigated the impacts of forest gaps on fruit-feeding butterflies in the Sabangau peat-swamp forest, Central Kalimantan, Indonesia. Fruit-baited traps were used to monitor butterflies for 3 months during the 2009 dry season. A network of 34 traps (n_gap = 17, n_shade = 17) was assembled in a grid covering a 35 ha area. A total of 445 capture events were recorded, comprising 384 individuals from 8 species and 2 additional species complexes classified to genera. On an inter-site scale, canopy traps captured higher species richness than understory traps; however, understory traps captured higher diversity within each site. Species richness was positively correlated with percent canopy cover and comparisons of diversity indices support these findings. Coupled with results demonstrating morphological differences in thorax volume and forewing length between species caught in closed-canopy traps vs. those in gaps, this indicates that forest degradation has a profound effect on butterfly communities in this habitat, with more generalist species being favored in disturbed conditions. Further studies are necessary to better understand the influences of macro-habitat quality and seasonal variations on butterfly diversity and community composition in South-east Asian peat-swamp forests.     

Cyclestheria hislopi (Crustacea: Branchiopoda): A group of morphologically cryptic species with origins in the Cretaceous

Cyclestheria hislopi is thought to be the only extant species of Cyclestherida. It is the sister taxon of all Cladocera and displays morphological characteristics intermediate of Spinicaudata and Cladocera. Using one mitochondrial (COI) and two nuclear (EF1α and 28S rRNA) markers, we tested the hypothesis that C. hislopi represents a single circumtropic species. South American (French Guiana), Asian (India, Indonesia, Singapore) and several Australian populations were included in our investigation. Phylogenetic and genetic distance analyses revealed remarkable intercontinental genetic differentiation (uncorrected p-distances COI > 13%, EF1α > 3% and 28S > 4%). Each continent was found to have at least one distinct Cyclestheria species, with Australia boasting four distinct main lineages which may be attributed to two to three species. The divergence of these species (constituting crown group Cyclestherida) was, on the basis of phylogenetic analyses of COI and EF1α combined with molecular clock estimates using several fossil branchiopod calibration points or a COI substitution rate of 1.4% per million years, dated to the Cretaceous. This was when the South American lineage split from the Asian–Australian lineage, with the latter diverging further in the Paleogene. Today’s circumtropic distribution of Cyclestheria may be best explained by a combination of Gondwana vicariance and later dispersal across Asia and Australia when the tectonic plates of the two continents drew closer in the early Miocene. The lack of morphological differentiation that has taken place in this taxon over such a long evolutionary period contrasts with the high level of differentiation and diversification observed in its sister taxon the Cladocera. Further insights into the evolution of Cyclestheria may help us to understand the evolutionary success of the Cladocera.     

Applicability of massively parallel sequencing on monitoring harmful algae at Varna Bay in the Black Sea

In this study the plankton diversity in 13 environmental samples from Varna Bay (in the western Black Sea) was analyzed using massively parallel sequencing (MPS). This preliminary study was undertaken to assess the potential of this technology for future implementation in monitoring programs in the Black Sea. Amplicon sequences of the 18S rRNA gene (V4-5 regions) were obtained using the Illumina MiSeq 250PE platform. A total of 1137 operational taxonomic units (OTUs) were obtained among which 242 OTUs with >0.990 BLAST top hit similarity (21.3% of all detected OTUs) closely related to sequences belonging to −protists. A large portion (175 OTUs = 72.3%) was identified at the species levels, including species typical for the Bulgarian Black Sea plankton community, as well as many that haven’t been reported earlier in the Bulgarian Black Sea coast (124 OTUs = 51.2%). Dinoflagellates were represented by the highest species number (77 OTUs comprising 31.8% of protist species), with dominant genera Gyrodinium and Heterocapsa. The present survey revealed the presence of 12 species listed as harmful, some of which have been previously overlooked, such as Cochlodinium polykrikoides, Karenia bicuneiformis, and Karlodinium veneficum. Species identification was possible for 10.3–36.0% of the detected OTUs in the six major supergroups. The frequency in Rhizaria was significantly lower than that in other major groups (p < 0.05–0.01), implying difficulties in the classification from morphology-based observations. The metagenetic data had an insufficient resolution of the 18S rRNA gene for species identification in many genera. These issues may hamper the implementation of MPS-based surveys for plankton monitoring, especially for detecting harmful algal blooms (HAB). The sequencing technology is steadily improving and it is expected that sequence length and quality issues will be resolved in the near future. The ongoing efforts to register taxonomic information and quality controls in the international nucleotide sequence databases (INSDs) will be essential for improving taxonomic identification power.     

DNA barcoding species in Alexandrium tamarense complex using ITS and proposing designation of five species

Alexandrium species can be very difficult to identify, with A. catenella, A. tamarense, and A. fundyense that compose “Alexandrium tamarense species complex” (Atama complex) as a distinct example. DNA barcoding is promising to offer a solution but remains to be established. In this study, we examined the utility of ITS in resolving the Atama species complex, by analyzing previously studied strains plus unstudied Chinese strains within the LSU- and SSU-rDNA based group/clade frameworks recently established. We further investigated the presence of intragenomic polymorphism and its implications in species delimitation. Similar to the previous SSU and LSU results, our ITS-based phylogenies divided the complex to five clusters, but with longer and evener branch lengths between the clusters. Based on the ITS region, the inter-cluster genetic distances (p = 0.134–0.216) were consistently and substantially greater than intra-cluster genetic distances (p = 0.000–0.066), with an average inter-cluster (species) distance (p = 0.167) 7.6-fold of the average intraspecific difference (p = 0.022), qualifying the approximately 510–520 bp ITS as a DNA barcode for Atama complex. We detected varying levels of intragenomic polymorphism in ITS but found that this did not impact the taxon-resolving power of this gene. With this DNA barcode, the new East and South China Sea strains and one Antarctic strain were placed in Clade IIC/Group IV, even though there were 7–10 polymorphic sites in their ITS, in contrast to none in SSU. Furthermore, our results suggest that the five clusters are recognizable as distinct species according to the phylogenetic species concept. Based on the phylogenetic placements of the type-locality strains of the existing three morphospecies and the dominant localities of other strains, we propose that Group I/Clade I be designated as A. fundyense, Group III/Clade IIB as A. tamarense, Group IV/Clade IIC as A. catenella, Group II/Clade IIA as A. mediterranis, and Group V/Clade IID as A. australis.     

High allozymic diversity in natural populations of Mycoheterotrophic Orchid Gastrodia elata,an endangered medicinal plant in China

Gastrodia elata Blume, a mycoheterotrophic orchid native to the Far East, is an endangered medicinal plant in China. Genetic variation among 19 natural populations of G. elata was examined in central China by using allozyme polymorphism (16 loci in six enzymatic systems). The species exhibited high level of genetic diversity (P = 56.3%, A = 2.2 and H_E = 0.221), which was mainly attributed to its perennial habit and mixed reproduction system (both sexual and asexual). Evident genetic differentiation in G. elata natural populations was suggested by F_ST = 0.241. AMOVA analysis showed 31.3% of the total molecular variation was attributed to inter-population differentiation. Obvious genetic structure and genetic depauperation of some populations indicate forest fragmentation and over-collection have affected genetic variation of G. elata. A conservation strategy, which is conserving populations with great genetic distinction or high level of genetic variation from four management units, is recommended.     

Genetic structure of Octopus vulgaris (Cephalopoda,Octopodidae) in the central Mediterranean Sea inferred from the mitochondrial COIII gene

The polymorphism of the mitochondrial gene cytochrome oxidase III was studied in the Mediterranean octopus, Octopus vulgaris Cuvier, 1797. A total of 202 specimens from seven sampling sites were analysed with the aim of elucidating patterns of genetic structure in the central Mediterranean Sea and to give an insight into the phylogeny of the Octopus genus. Phylogenetic analyses showed that individuals from the central Mediterranean belong to the O. vulgaris species whose limits should nevertheless be clarified. Concerning genetic structure, two high-frequency haplotypes were present in all locations. The overall genetic divergence (Φ_ST = 0.05, P < 0.05) indicated a significant genetic structuring in the study area and an AMOVA highlighted a significant break between western and eastern Mediterranean basins (Φ_CT = 0.094, P < 0.05). Possible explanations for the observed patterns of genetic structuring are discussed with reference to their relevance for fisheries management.     

Phylogenetic structure of the Thomomys bottae–umbrinus complex in North America

The phylogeography of the Thomomys bottae–umbrinus complex in the United States and Mexico was assessed with sequences of the mitochondrial cytochrome b gene. These sequences were obtained from 225 individuals representing 108 locations over the range, including 56 sequences from GenBank. 110 (500 bp) sequences were used for Bayesian inference and neighbor-joining analyses, and 34 (1140 bp) specimens from the main clades obtained from the Bayesian inference were used in maximum-parsimony and maximum-likelihood analyses. The different analyses indicate significant variation within the species complex that averages 13% among major groups of genetic differences among Thomomys bottae–umbrinus. The overall pattern of geographic variation is not concordant with the current taxonomy. To the contrary, eight monophyletic groups are supported by all analyses and can be considered phylogenetic species. Overall divergence among these groups appears influenced by historical biogeographic events active during the Pliocene and Pleistocene.     

Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis

Studies on the microbial ecology of gut microbiota in bats are limited and such information is necessary in determining the ecological significance of these hosts. Short-nosed fruit bats (Cynopterus brachyotis brachyotis) are good candidates for microbiota studies given their close association with humans in urban areas. Thus, this study explores the gut microbiota of this species from Peninsular Malaysia by means of biochemical tests and 16S rRNA gene sequences analysis. The estimation of viable bacteria present in the stomach and intestine of C. b. brachyotis ranged from 3.06 × 10¹⁰ to 1.36 × 10¹⁵ CFU/ml for stomach fluid and 1.92 × 10¹⁰ to 6.10 × 10¹⁵ CFU/ml for intestinal fluid. A total of 34 isolates from the stomach and intestine of seven C. b. brachyotis were retrieved. A total of 16 species of bacteria from eight genera (Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas and Serratia) were identified, Enterobacteriaceae being the most prevalent, contributing 12 out of 16 species isolated. Most isolates from the Family Enterobacteriaceae have been reported as pathogens to humans and wildlife. With the possibility of human wildlife transmission, the findings of this study focus on the importance of bats as reservoirs of potential bacterial pathogens.     

Fungal endophyte–grass symbioses are rare in the California floristic province and other regions with Mediterranean-influenced climates

Research on agronomic grasses has shown that Class 1 fungal endophytes (Neotyphodium/Epichloë; Clavicipitaceae) can have profound effects on host plant fitness. However, in natural systems, even basic ecological knowledge of most endophyte symbioses is lacking. Here, I describe the distribution and abundance of endophytes across 36 native (or naturalized) grasses in a previously unsurveyed region, the California Floristic Province. Symbiosis was generally low: 8.33 % of species and 18.75 % of genera hosted endophytes. I then compared the proportions of symbiotic species and genera found in California and other Mediterranean regions to the proportions found in non-Mediterranean regions. Surveys of Mediterranean-influenced regions showed significantly lower proportions of species (～66 % lower) and genera (～65 % lower) hosting endophyte than surveys of non-Mediterranean regions. This pattern suggests that selection in Mediterranean climates may not favor endophyte symbioses.     

Taxonomic diversity and community structure of arbuscular mycorrhizal fungi (Phylum Glomeromycota) in three maritime sand dunes in Santa Catarina state,south Brazil

Community structure and species richness of arbuscular mycorrhizal fungi (Phylum Glomeromycota) were studied in sand dune sites at Itapiruba (southern), Joaquina (intermediate) and Praia Grande (northern) beaches along the coast of the state of Santa Catarina, Brazil. In each site, a 20 × 20 m plot was established and 20 soil samples collected in a regular grid pattern. Fungal spores were extracted from each sample, counted and identified to species level. A total of 25 species were recovered belonging to seven genera and five families in the Glomeromycota. Gigaspora albida and Acaulospora scrobiculata occurred in >50 % of samples at all three sites. Other common species whose sample frequency was >50 % in one or two sites were Scutellospora weresubiae, Scutellospora cerradensis and Racocetra gregaria, while the remaining majority of species were detected in <25 % of samples within a given site. Dune sites could be differentiated based on the higher frequency of occurrence of S. cerradensis and Acaulospora morrowiae in Itapiruba, S. weresubiae in Joaquina, and Scutellospora hawaiiensis in Praia Grande. No differences across sites were observed for species richness and total spore numbers, the latter averaging from 28.8 to 31.8 spores per 100 ml soil. Shannon diversity was significantly higher in Praia Grande compared to the other two sites. Differences in the relative spore abundance of genera among dunes were detected only for Scutellospora, which was significantly more abundant in the Joaquina beach. Community structure, as depicted by species rank/log abundance graphs, was not significantly different between areas according to the Kolmogorov–Smirnov two-sample test. Species accumulation curves demonstrated that 13 samples were enough to detect 90 % of all species. Overall, sand dune systems share similar arbuscular mycorrhizal fungal communities despite being geographically distant (150 km) from each other.     

Genetic studies in the recently divergent Eligmodontia puerulus and E. moreni (Rodentia,Cricetidae, Sigmodontinae) from Puna and Monte deserts of South America

Eligmodontia is a genus of phyllotine rodents adapted to arid environments with seven recognized species. The sister species E. puerulus and E. moreni are distributed in the adjacent highland Puna and lowland Monte deserts respectively, and show remarkable morphological and chromosomal differences. However, analyses of the cytochrome b gene showed important variability, without reciprocal monophyly between them. In order to study the evolutionary processes involved in the diversification of both taxa, we analyzed 1161 bp of the mitochondrial control region and flanking sequences (N = 60), as well as 759 bp of the first exon of the nuclear gene IRBP (N = 14). Individuals of both species from Jujuy, Catamarca and Mendoza Provinces of Argentina were previously karyotyped. Results showed that the mitochondrial sequences present high haplotype and nucleotide diversity within all population, and no haplotype was shared between both species. F_ST indicated that populations of both species were moderately structured. The network was constituted by two major haplogroups, one composed by E. puerulus samples from Jujuy, and the other composed of sequences of all studied populations. The Bayesian analysis showed three clusters, matching the network. Phylogenetic analysis recovered two clades with high support, in coincidence with the network groups. There was only one close join between sequences of both species, corresponding to samples from Catamarca. Thus, mitochondrial data suggested hybridization between both species in Catamarca, with asymmetric introgression. The IRBP showed low variability and, in the phylogenetic analysis, the sequences of E. puerulus form a monophyletic group with intermediate support, whereas those of E. moreni collapse into a basal polytomy. Our data indicated a recent divergence and absence of introgression in the nuclear genomes. The results at the population level with mitochondrial sequences, together with integrative taxonomy at the species level in a biogeographic context, suggest that climatic and geologic changes could have had an important role in the determination of genetic variability patterns observed in these rodents.     

Extinction thresholds and negative responses of Afrotropical ant-following birds to forest cover loss in oil palm and agroforestry landscapes

Afrotropical ant-following birds are vulnerable to forest loss and disturbance, but critical habitat thresholds regarding their abundance and species richness in human-dominated landscapes, including industrial oil palm plantations, have never been assessed. We measured forest cover through Landsat imagery and recorded species richness and relative abundance of 20 ant-following birds in 48 plots of 1-km², covering three landscapes of Southwest Cameroon: Korup National Park, smallholder agroforestry areas (with farms embedded in forest), and an industrial oil palm plantation. We evaluated differences in encounter frequency and species richness among landscapes, and the presence of critical thresholds through enhanced adaptive regression through hinges. All species were detected in Korup National Park and the agroforestry landscape, which had similar forest cover (>85%). Only nine species were found in the oil palm plantation (forest cover = 10.3 ± 3.3%). At the 1-km² scale, the number of species and bird encounters were comparable in agroforests and the protected area: mean species richness ranged from 12.2 ± 0.6 in the park and 12.2 ± 0.6 in the agroforestry matrix to 1.0 ± 0.4 in the industrial oil palm plantation; whereas encounters decreased from 34.4 ± 3.2 to 26.1 ± 2.9 and 1.3 ± 0.4, respectively. Bird encounters decreased linearly with decreasing forest cover, down to an extinction threshold identified at 24% forest cover. Species richness declined linearly by ca. one species per 7.4% forest cover lost. We identified an extinction threshold at 52% forest cover for the most sensitive species (Criniger chloronotus, Dicrurus atripennis, and Neocossyphus poensis). Our results show that substantial proportions of forests are required to sustain complete ant-following bird assemblages in Afrotropical landscapes and confirm the high sensitivity of this bird guild to deforestation after industrial oil palm development. Securing both forest biodiversity and food production in an Afrotropical production landscape may be best attained through a combination of protected areas and wildlife-friendly agroforestry.