16S mtDNA Research Articles

Optimization of Wetland Environmental DNA Metabarcoding Protocols for Great Lakes Region Herpetofauna

ABSTRACTMany species of reptiles and amphibians (herpetofauna) rely on wetlands that are being degraded and lost at a high rate. Characterization of herpetofauna diversity in different wetland types may help guide conservation strategies. However, traditional survey methods often involve sampling within small temporal windows, and the gear deployed may be taxonomically biased, thus, they may fail to accurately characterize species presence/absence and diversity. In contrast, environmental (e)DNA metabarcoding has been shown to effectively survey entire aquatic communities and can provide a useful complement to traditional surveys. The objective of this study was to design and optimize eDNA sampling and laboratory protocols for wetland herpetofauna. Protocols evaluated included different water sampling approaches (point versus transect sampling), seasonality of sampling, and choice of metabarcoding marker (mitochondrial 12S versus 16S rDNA). Samples collected from 10 sites across southern Michigan detected 17 amphibian and five reptile species, including four species of conservation concern (Ambystoma texanum, Clemmys guttata, Rana palustris, and Sternotherus odoratus). We observed no difference in the number of species detected between point and transect samples (p = 0.70), but point sampling required less time (p = 0.03) and allowed significantly larger volumes of water to be filtered (p = 1.13e‐5). No difference in species richness was observed between the 12S and 16S mitochondrial DNA markers (p = 0.96). However, a greater number of taxa were identifiable at the species level when using the 16S locus. There was also a significant difference in the number of species detected between early and late summer sampling periods (more species detected in the earlier period; p = 6.31e‐6), and some species were only found in the early or late sampling period. Sampling during multiple periods to fully characterize species composition, the use of point sampling, and the 16S mtDNA marker for herpetofauna eDNA metabarcoding studies may increase efficiency and reliability of results.

Molecular identification of goat lice (Anoplura: Linognathidae) based on conserved motif analysis

Goat lice (Anoplura: Linognathidae) are important ectoparasites in pediculosis. However, their molecular classification and identification remain challenging owing to a lack of molecular data in GenBank. This study aimed to establish a molecular method for identifying Anoplura spp. Goat lice were captured in Yulin, China, and morphologically identified to Linognathus africanus (L. africanus). Sequences across Anoplura were downloaded from GenBank and conserved motif analysis showed that ribosomal DNA (rDNA) 18S V4, mitochondria DNA (mtDNA) 12S, and 16S were candidate gene fragments suitable for universal primer design because of abundant sequences and long conserved motifs with few mutations. These three gene fragments of the lice specimens were successfully amplified and sequenced, and their divergences were 0.1–1.7%, 0–0.6%, and 0.3–1.3%, respectively, indicating that the lice specimens belonged to the same species. BLAST analysis showed that the 18S sequences were only aligned to Linognathus, while the 12S and 16S sequences showed 98.8–99.4% and 98.7–99.3% similarities, respectively, with those of L. africanus from Pakistan. Therefore, the lice specimens were molecularly identified as L. africanus without geographical isolation. In conclusion, the goat lice specimens were identified to L. africanus. 12S and 16S are potential DNA barcodes of Anoplura spp.

A molecular approach to identify parrotfish (Sparisoma) species during early ontogeny.

Sparisoma species (parrotfish) comprise an important functional group contributing to coral-reef resilience. The morphological diagnostic characteristics for species identification are clearly described for adult forms but not for the early stages. Consequently, many taxonomical listings of Sparisoma larvae are restricted to the genus level. The aims of this study are to determine whether the morphological and molecular identification techniques are useful to assign the species taxonomic level to Sparisoma larvae occurring in the Gulf of Mexico and whether there is a set of diagnostic features that could be used to discriminate between species in larvae of different developmental stages. Morphological assignment of Sparisoma was performed based on morphological and meristic features for 30 larvae collected in the Gulf of Mexico from late August to mid-September 2015. To corroborate and complement the morphological assignments, molecular identification was carried out using DNA sequences from regions of two mitochondrial genes, mitochondrial cytochrome oxidase I (mtDNA COI) and mitochondrial 16S rRNA (mtDNA 16S rRNA). COI and 16S gene trees for Sparisoma and related fish taxa were constructed using sequences available in the NCBI (National Center for Biotechnology Information) GenBank and BOLD (Barcode of Life Data) databases. Two morphotypes were identified based on morphology, but no diagnostic characteristics for species discrimination were found. Molecular identification, in contrast, successfully discriminated four early development stages of Sparisoma atomarium, three stages of Sparisoma radians, and two stages of Sparisoma chrysopterum and Sparisoma aurofrenatum, therefore demonstrating the successful and necessary application of molecular taxonomic approaches for species-level identifications of Sparisoma larvae.

Identification of Fish Species Involved with Ciguatera Food Poisoning in Okinawan Waters by Using PCR-RFLP analysis

Ciguatera fish poisoning (CFP), known as a seafood-borne disease, is caused by consumption of fish contaminated with ciguatoxins in tropical and subtropical sea. The ciguatera fishes, Variola louti, Lutjanus monostigma and L. bohar have an absolute majority in the Ryukyu Archipelago, southwestern Japan. We developed the cluster analysis of phylogenetic tree by using mitochondrial (mt) DNA 16S rRNA sequences of V. louti, L. monostigma and L. bohar and differentiate them from morphologically similar species (L. fulviflamma, L. russellii, L. argentimaculatus, Plectropomus leopardus and V. albimarginata) in our previous study. The fish were acquired from the coastal waters of the Ryukyu Archipelago, and a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) marker of the mtDNA 16S rRNA region was used, employing the restriction enzymes BmgT120 I, Dde I, and SnaB I, to identify the fish species responsible for CFP. These results showed that a PCR-RFLP marker can be obtained more easily than a nucleotide sequence.

Molecular phylogeny and taxonomic review of the family Pomacentridae from Korea

A total of eight genera and 21 species of the family Pomacentridae have been reported in Korea, but the classification is uncertain. Therefore, this study aims to clarify molecular phylogeny and classification of eight genera and 17 species of the family Pomacentridae from Korea based on mtDNA sequences (16S rRNA 473 bp), nDNA sequences (RAG1 812 bp) and morphological traits. Molecular phylogenetic trees (ML, and BI) showed that four groups (Chrominae, Glyphisodontinae, Pomacentrinae, and Microspathodontinae) were largely formed, and monophyletic relationship was observed four groups. Each subfamily is divided by the number of spiniform procurrent caudal rays, the shape and rows of the teeth, and the margin of suborbital and preopercle. Each genus is divided by the number of dorsal fin spines and anal fin soft rays, the shape and rows of the teeth, and body coloration. Through an extensive examination of pomacentrids specimens loaned from museum or universities, it is believed that Chromis analis, Neopomacentrus violascens and Pomacentrus chrysurus are not distributed in Korea in spite of their previous records. Finally, we provide key to the species of the family Pomacentridae from Korea.

Environmental DNA metabarcoding on aquatic insects: Comparing the primer sets of MtInsects‐16S based on the mtDNA 16S and general marker based on the mtDNA COI region

AbstractLong‐term biodiversity monitoring is necessary for conservation and management. In such circumstances, environmental DNA (eDNA) surveys can enable easy and effective biomonitoring of aquatic insects. However, previous studies of aquatic insects based on the mtDNA COI region have revealed incomplete taxonomic coverage, and frequent amplification of nontarget taxa (e.g., algae and diatoms). Additionally, it has been indicated that there are few reference sequences registered in databases for metabarcoding analysis of insects. Therefore, we developed a new primer set, MtInsects‐16S, for eDNA analyses of insects based on the mtDNA 16S rRNA region in a previous study. To address the insufficient database records of insect DNA sequences, we also constructed a comprehensive reference database of aquatic insects that occur in Kanagawa Prefecture, Japan. We conducted eDNA analyses at six sites in the two river systems (the Sagami‐gawa River and Sakawa‐gawa River systems), with three replicates per site, based on both the mtDNA COI and the 16S rRNA regions. These results were compared physical capture surveys at the same sites to examine the detection capability of eDNA for Insecta with using a well‐established database. Among the list of species which were collected by physical capture surveys, 74.9% were detected by MtInsects‐16S, whereas 40.1% were detected by the primer set of the mtDNA COI region, and also 80.0% were detected by the both primer sets. This study demonstrated that the application of eDNA analyses using the MtInsects‐16S primer set can be conducted with accuracy and reliability, provided that the reference DNA database is improved. The MtInsects‐16S region can be considered indispensable in eDNA analysis for aquatic insects.

Detecting kelp-forest associated metazoan biodiversity with eDNA metabarcoding

Environmental DNA (eDNA) metabarcoding is a promising tool for monitoring marine biodiversity, but remains underutilised in Africa. In this study, we evaluated the ability of aquatic eDNA metabarcoding as a tool for detecting biodiversity associated with a South African kelp forest, an ecosystem that harbours high diversity of species, many of which are endemic, but are also sensitive to changing environmental conditions and anthropogenic pressures. Using fine-scale spatial (1 m and 8 m) and temporal (every four hours for 24 h) sampling of aquatic environmental DNA and targeting two gene regions (mtDNA COI and 12S rRNA), metabarcoding detected 880 OTUs representing 75 families in the broader metazoan community with 44 OTUs representing 24 fish families. We show extensive variability in the eDNA signal across space and time and did not recover significant spatio-temporal structure in OTU richness and community assemblages. Metabarcoding detected a broad range of taxonomic groups, including arthropods, ascidians, cnidarians, echinoderms, ctenophores, molluscs, polychaetes, ichthyofauna and sponges, as well as Placozoa, previously not reported from South Africa. Fewer than 3% of OTUs could be identified to species level using available databases (COI = 19 OTUs, 12S = 11 OTUs). Our study emphasizes that kelp-forest associated biodiversity in South Africa is understudied, but that with careful consideration for sampling design in combination with increased barcoding efforts and the construction of regional databases, eDNA metabarcoding will become a powerful biomonitoring tool of kelp-forest associated biodiversity.

A new species of Vietnamophryne (Anura: Microhylidae) from Northeastern Vietnam.

A new species of the genus Vietnamophryne is described from Vietnam on the basis of two specimens collected from Tuyen Quang Province, Northeastern Vietnam. The new species is morphologically most similar to Vietnamophryne occidentalis from Thailand, however, it differs from the latter by having large black blotches in the lower jaw region, and a yellow-orange chest and belly. The genetic distance between the new species and other Vietnamophryne taxa is > 2.13% (16S mtDNA gene fragment). Vietnamophryne aurantifusca sp. nov. can be distinguished from all other species of Vietnamophryne by a combination of the following morphological characteristics: Size medium (SVL 17.618.2 mm in males); head wider than long; tympanum medium; finger I longer than half of finger II; dorsal skin relatively smooth with some round nodules, concentrated in the middle of the back, arranged along the length of the back, with a prominent ridge along the spine; Dorsum orangish-brown entirely and paler on margin of back with a small brownish ridge along the spine; sides brownish with creamy patches and orange spots; ventral surface orange, with grey marbling, most intense on the throat, ventral side of arms and thighs, and ventral surfaces of limbs dark grey with some orange spots.

Phylogeography of an insect inhabiting ‘Sky Islands’: the relationships among genetic structures and geographical characteristics, geohistorical characteristics, and cyclical climate changes

Abstract When gene flow has been restricted between populations, the genetic structure of such species often reflects geohistory and climate changes. Populations of species inhabiting high-altitude regions, known as ‘Sky Islands’, are isolated and exhibit restricted gene flow, so they often have habitat-specific genetic structures that correspond to their surrounding geographical structures. Here we focus on a limnephilid caddisfly, Rivulophilus sakaii, which inhabits the alpine zone of Japan. Phylogenetic analyses were conducted based on the mtDNA COI and 16S rRNA regions, and the nDNA 18S rRNA, 28S rRNA, CAD, EF1-α, and POL-II regions; the results indicated three phylogeographically differentiated intraspecific lineages. Haplotype network and demographic analyses based on the mtDNA COI region suggested the size of the respective isolated populations has stabilized. This suggests that mountain formation in the Japanese Archipelago due to volcanic activity has resulted in barriers to migration and dispersal between high-altitude aquatic insect populations. This was inferred to be an effect of Quaternary climate changes that caused vertical distributional shifts following mountain formation, resulting in repeated connection and fragmentation of the populations. This is important supporting information with regard to discussing the effects and functions of geohistory and climatic changes on the phylogenetic evolution of organisms presently inhabiting interglacial ‘Sky Islands’.

Environmental DNA biomonitoring in biodiversity hotspots: A case study of fishes of the Okavango Delta

AbstractThe Okavango Delta is the largest freshwater wetland in southern Africa and a recognized biodiversity hotspot and UNESCO World Heritage Site. The region is extremely rich in floral and faunal diversity, including a fish fauna of ~90 species in 15 families, that also support recreational and subsistence fishing. Anthropogenic pressures and invasive species threaten the unique biodiversity and ecosystem services that the Delta provides, necessitating biomonitoring tools that can provide broad community‐level diversity insights. Here, we utilize environmental DNA metabarcoding of aquatic eDNA using the MiFish 12S rRNA primers, to investigate fish communities and also sequenced 211 mtDNA 12S barcodes for 74 species across 36 genera of fishes from the region. Metabarcoding recovered 11 of 15 families, with 40 species detected across 23 genera, representing ~50% of known diversity, with the mtDNA 12S fragment able to delineate all genera (except for the cichlid genera Serranochromis and Pharyngochromis that comprised a single clade) and most species, except for some in the Clarias, Enteromius, Labeo, Lacustricola, and Petrocephalus genera. Generally, abundant and wide‐spread taxa such as Clarias spp. and Marcusenius altisambesi, amongst others, were often detected in the surveys, with other species, including Zaireichthys kavangoensis, Schilbe intermedius, and Labeo sp. detected less frequently. Dissolved oxygen, temperature, and dissolved organic solids were positively correlated with community diversity, highlighting the influence of environmental factors in shaping fish communities in the region. Further, there was strong variability in the eDNA signal across only 1000 m, suggesting that future surveys need to consider spatio‐temporal aspects of sample collection. Our study highlights the potential of eDNA metabarcoding for surveying aquatic biodiversity in the Okavango Delta, particularly within the context of baseline biodiversity inventories, that underpin conservation and management initiatives. As such, we provide a number of recommendations that can help structure future sampling efforts in the region.

Revision of the Subgenus Ochthomantis Frogs from Madagascar (Amphibia: Mantellidae) with the Description of Four Species and Resurrection of Mantidactylus catalai and M. poissoni.

The subgenus Ochthomantis is an obligate forest and stream-dwelling group of mantellid frogs, endemic to Madagascar, with six species currently recognized. However, this group suffers from ongoing taxonomic confusion due to low numbers of examined specimens, and failure to consider morphological variation from development and sexual dimorphism. Here, we examined the morphology of 637 sexed adult specimens collected by us in the field and from other museum collections. We also sequenced a DNA fragment of the 16S mtDNA gene for each lineage to determine congruence between morphological and molecular data sets and to help delimit species. Our results demonstrate that the subgenus Ochthomantis includes eleven valid species: five already recognized, M. catalai and M. poissoni that we resurrect from synonymy, and four new species which we describe for the first time here. In some analyses, Mantidactylus majori groups with other Mantidactylus subgenera, so we do not consider it a member of the subgenus Ochthomantis in this study. All species have restricted distributions and elevational ranges in the humid forests of Madagascar. This study demonstrates the utility of assessing cryptic species using both diagnostic morphological characters and molecular data. The discovery of this new cryptic biodiversity, and the taxonomic revision herein, will likely require conservation activities for those species with the most restricted distributions.

A new species of the genus Leptobrachella Smith 1925 (Amphibia, Anura, Megophryidae) from Guangxi, China.

A new species of Leptobrachella, L.wumingensissp. nov., was described from the Damingshan National Nature Reserve, Wuming District, Nanning City, Guangxi, China based on morphological, molecular and bioacoustic data. Phylogenetic analysis of 16S mtDNA fragments revealed that the new species is closely related to L.damingshanensis. Uncorrected p-distances between the new species and all homologous DNA sequences available for the 16S gene of Leptobrachella are greater than 7.1%. Morphologically, L.wumingensissp. nov. differs from its congeners in several ways, including a medium body size (SVL 26.0-26.7 mm in males, 30.6-34.8 mm in females), lack of toe webbing and lateral fringes, shagreened and granular dorsal surface, pale brown dorsum with darker brown markings, iris bicolored, with the upper half copper and fading to silver in the lower half, and the presence of small irregular black spots and tangerine tubercles on the flanks. Furthermore, we found the new species to have two types of advertisement calls and relatively high dominant frequencies, making it distinct from its congeners.

Haplotype and phylogenetic diversity using mitochondrial 12S rRNA gene marker in Bali cattle

Bali cattle is one of Indonesia's original livestock genetic resources. The 12S rRNA can be used as a marker of genetic diversity, and until now, there has been no report on Bali cattle. The study was aimed to analyze the gene of mtDNA 12S rRNA to determine haplotype diversity and phylogenetics in Bali cattle populations of the eastern region of Indonesia. A total of 95 blood samples used consisted of three different populations which were Bali cattle, Ongole crossbred (PO) and Madura cattle as a comparison. This research was analyzed using PCR and sequencing methods. The data were analyzed using the cluster W method for estimating genetic distances, calculating diversity, and reconstructing phylogenetic trees using the MEGAX, DNAsP and Network. The genetic distance values ranged from 0.00200 to 0.01508, and the Haplotype diversity values ranged from 0.66000 to 0.91111. The nucleo-tide diversity values ranged from 0.00174 to 0.01673. There were 16 haplotypes found. The values of Gst, Nst and Fst were 0.00803, 0.07550, 0.07622; respectively. Based on the analysis, there were dif-ferences between Bali cattle from various populations, there were specific haplotypes. The 12S rRNA gene can be used as a genetic marker for diversity studies in Bali cattle and other cattle breeds although the diversity is low.

Considerable gene flow in troglomorphic cockroach species across a vast subterranean landscape

AbstractAimThere has been growing interest in non‐cave subterranean habitats and their influence on the evolution of troglomorphic (i.e. ‘subterranean adapted’) species. Studies on the diversification of aquatic subterranean organisms in these habitats generally support the ‘subterranean island’ hypothesis, whereby isolated subterranean refuges lead to patterns of short‐range endemism. However, their terrestrial counterparts have received less attention. We aimed to elucidate the applicability of the ‘subterranean island’ hypothesis to terrestrial subterranean fauna through genetic analyses of two widespread troglomorphic cockroach species. To investigate the influence of subterranean biogeography, we also analysed a closely related species that inhabits ‘classic’ cave environments to represent a contrasting biogeographic comparison.LocationPilbara region, Western Australia, and the Chillagoe‐Mungana Caves, Queensland (Australia).TaxaCave cockroach species: Nocticola cockingi, Nocticola quartermainei and Nocticola australiensis.MethodsWe used DArTseq to generate genome‐wide SNPs in 78 samples, and Sanger sequencing to generate 16S mtDNA data. We then applied various population genomic analyses to characterize the distribution of genetic diversity within the three study species.ResultsWe identified distinct genetic clusters within the two Pilbara species; however, there appeared to be a notable lack of discernible population differentiation across large parts of their range (>135 km), opposing the subterranean island hypothesis. The highest level of population differentiation in the three study species was between the two caves in Queensland, ~3 km apart.Main ConclusionsThe Pilbara subterranean habitat appeared to be conducive to gene flow across relatively large distances, contrasting high levels of endemism observed in other subterranean taxa within the region. The disparate patterns of gene flow among the Pilbara and Queensland study species emphasize the significance of differing subterranean habitats on patterns of dispersal and vicariance. These inferences will inform conservation genetic management of these species, and may help elucidate the evolutionary paradox of widespread subterranean fauna.

Phylogeography of the true freshwater crab, Geothelphusa dehaani: Detected dual dispersal routes via land and sea

Dispersal is an important factor that determines the potential for colonization to pioneer sites. Although most decapods employ seaward migration for reproduction with a planktonic larval phase, true freshwater crabs spend their entire life cycle in freshwater. Therefore, it is expected that genetic regionality can be easily detected. In this study, we focused on true freshwater crabs, Geothelphusa Stimpson, 1858. Herein, we reveal the evolutionary history and dispersal patterns of freshwater crustaceans. We collected and genetically analyzed 283 specimens at 138 localities across the Japanese Islands. Phylogenetic analyses were conducted on the combined dataset (mtDNA COI, 16S, and nDNA ITS1, histone H3 regions) and the data set based on the mtDNA COI region. The phylogenetic relationships detected 10 clades that were highly monophyletic. The highlights of this study were the discovery of several cryptic species or undescribed species, and the completely different heterogeneous dual dispersal pathways within a single species; i.e., both land and ocean routes. Although it was concluded that Japanese crabs are basically genetically divided by straits, strong evidence for dispersion via ocean currents was also detected (i.e., a “sweepstake”). It was also confirmed that Geothelphusa dehaani (White, 1847) could survive in seawater.

Evaluation of commercial meat product food label conformity using multiplex PCR assay

This study evaluated the authenticity of pre-packaged processed beef products using multiplex PCR (mPCR) analysis on mtDNA. A total of 48 commercial beef products, including meatballs, burgers, corned beef, smoked beef, and sausages, were examined. They were categorized into three price groups: low (<5 USD/kg), medium (5–10 USD/kg), and high (>10 USD/kg). The DNA genome was extracted and used to amplify mtDNA 12S rRNA to identify bovine, porcine, dog, and rat ingredients, as well as mtDNA cytochrome c oxidase subunit 1 (COI) to detect bovine, chicken, and porcine ingredients. The results showed that all samples contained bovine DNA (155 bp), based on mPCR 12S rRNA. Moreover, chicken DNA was detected in 33 beef products (596 bp) based on mPCR COI. Mislabeling was detected in 56.25% of beef products. In addition, chicken DNA was detected in all three price groups. In conclusion, some products did not comply with the label composition labeling, and higher prices did not guarantee beef product originality.

Validating the occurrence of the giant mottled eel (Anguilla marmorata) in the Galápagos Islands.

The giant mottled eel (Anguilla marmorata) is distributed mostly in the Indo-West Pacific. However, a few records indicate the presence of this eel in the Tropical Central and East Pacific. In April 2019, an eel specimen was caught in a small stream in San Cristobal Island, Galápagos. Morphological and molecular characters (16S and Cytb mtDNA sequences) confirmed the species as A. marmorata Quoy & Gaimard, 1824. The re-discovery of A. marmorata in Galápagos supports the hypothesis of an eastward range expansion from the west, probably through the North Equatorial Counter-Current.

Optimized concurrent hearing and genetic screening in Beijing, China: A cross-sectional study.

Concurrent screening has been proven to provide a comprehensive approach for management of congenital deafness and prevention of ototoxicity. The SLC26A4 gene is associated with late-onset hearing loss and is of great clinical concern. For much earlier detection of newborns with deafness-causing mutations in the SLC26A4 gene, the Beijing Municipal Government launched a chip for optimized genetic screening of 15 variants of 4 genes causing deafness based on a chip to screen for 9 variants of 4 genes, and 6 variants of the SLC26A4 gene have now been added. To ascertain the advantage of a screening chip including 15 variants of 4 genes, the trends in concurrent hearing and genetic screening were analyzed in 2019 and 2020. Subjects were 76,460 newborns who underwent concurrent hearing and genetic screening at 24 maternal and child care centers in Beijing from January 2019 to December 2020. Hearing screening was conducted using transiently evoked otoacoustic emissions (TEOAEs), distortion product otoacoustic emissions (DPOAE), or the automated auditory brainstem response (AABR). Dried blood spots were collected for genetic testing and 15 variants of 4 genes, namely GJB2, SLC26A4, mtDNA 12S rRNA, and GJB3, were screened for using a DNA microarray platform. The initial referral rate for hearing screening decreased from 3.60% (1,502/41,690) in 2019 to 3.23% (1,124/34,770) in 2020, and the total referral rate for hearing screening dropped form 0.57% (236/41,690) in 2019 to 0.54% (187/34,770) in 2020, indicating the reduced false positive rate of newborn hearing screening and policies to prevent hearing loss conducted by the Beijing Municipal Government have had a significant effect. Positivity according to genetic screening was similar in 2019 (4.970%, 2,072/41,690) and 2020 (4.863%,1,691/34,770), and the most frequent mutant alleles were c.235 del C in the GJB2 gene, followed by c.919-2 A > G in the SLC26A4 gene, and c.299 del AT in the GJB2 gene. In this cohort study, 71.43% (5/7) of newborns with 2 variants of the SLC26A4 gene were screened for newly added mutations, and 28.57% (2/7) of newborns with 2 variants of the SLC26A4 gene passed hearing screening, suggesting that a screening chip including 15 variants of 4 genes was superior at early detection of hearing loss, and especially in early identification of newborns with deafness-causing mutations in the SLC26A4 gene. These findings have clinical significance.

Unveiling the evolutionary relationships and the high cryptic diversity in Andean rainfrogs (Craugastoridae: Pristimantis myersi group).

Pristimantis is the most diverse genus of terrestrial frogs. Historically, it has been divided into several phenetic groups in order to facilitate species identification. However, in light of phylogenetic analysis, many of these groups have been shown to be non-monophyletic, denoting a high degree of morphological convergence and limited number of diagnostic traits. In this study, we focus on the Pristimantis myersi group, an assemblage of small rainfrogs distributed throughout the Andes of Ecuador and Colombia, whose external morphology is highly conserved, and its species diversity and evolutionary relationships largely unknown. We inferred a new phylogenetic hypothesis for the frog genus Pristimantis, including all available sequences of the mtDNA 16S rRNA, as well as new DNA sequences from 175 specimens. Our sampling included 19 of the 24 species currently recognized as part of the Pristimantis myersi group. Our new evolutionary hypothesis recovered the P. myersi group as non-monophyletic and composed of 16 species. Therefore, we exclude P. albujai, P. bicantus, P. sambalan, and P. nelsongalloi in order to preserve the monophyly of the group. We discovered at least eight candidate species, most of them hidden under the names of P. leoni, P. hectus, P. festae, P. gladiator, and P. ocreatus. Our results reveal the occurrence of a high level of cryptic diversity to the species level within the P. myersi group and highlight the need to redefine some of its species and reassess their conservation status. We suggest that the conservation status of six species within the group need to be re-evaluated because they exhibit smaller distributions than previously thought; these species are: P. festae, P. gladiator, P. hectus, P. leoni, P. ocreatus, and P. pyrrhomerus. Finally, given that the Pristimantis myersi group, as defined in this work, is monophyletic and morphologically diagnosable, and that Trachyphrynus is an available name for the clade containing P. myersi, we implement Trachyphrynus as a formal subgenus name for the Pristimantis myersi group.

DNA barcoding, dwelling morphology, and fecundity of the gall-forming shrimp Paratypton siebenrocki Balss, 1914 (Caridea: Palaemonidae)

ABSTRACT Tropical coral reefs offer a wide variety of habitats to countless invertebrate species. Sessile host organisms especially are inhabited by small taxa, of which decapod crustaceans form one of the most diverse communities. Symbiotic palaemonid shrimp species associate with marine invertebrate hosts from multiple phyla, including cnidarians such as stony corals (Scleractinia). The intriguing gall-forming shrimp Paratypton siebenrocki, a symbiont of Acropora corals in the Indo-Pacific, was collected in the Saudi Arabian Red Sea, Kenya, and the Maldives. Based on morphology P. siebenrocki has been considered to be most closely related to the genera Anapontonia and Metapontonia; however, no clear clustering with either palaemonid genus was observed in a phylogenetic reconstruction based on 16S and COI mtDNA. Here we photo-document the dwellings of P. siebenrocki in Acropora spp. for the first time, and furthermore we report on the reproductive output of this species. The number of eggs ranged from 345 to 909 (n = 6), and embryo volume differed strongly between early- and late-stage embryos. The carapace length ranged from 2.58 to 4.55 mm for the females and 1.51 to 2.5 mm for the males (n = 5). The number and size of the embryos, combined with their specialised, secluded lifestyle, suggest that P. siebenrocki allocates higher energy towards embryo production than free-living confamilials do.