Ethanol-preserved Samples Research Articles

Museomics reveal origins of East African Pleophylla forest chafers and Miocene forest connectivity

Here we present a nearly complete species-level phylogeny including 23 of the 25 known species of the forest-dwelling herbivorous scarab chafer beetle genus Pleophylla (Coleoptera: Scarabaeidae: Sericinae), based on the analysis of 950 nuclear genes (metazoan-level universal single-copy orthologs; mzl-USCOs). DNA sequences were obtained from freshly collected, ethanol-preserved samples and from dried museum specimens by target enrichment or genome shotgun sequencing. Alignment completeness of mzl-USCOs newly obtained here by target DNA enrichment of ethanol samples were very heterogenous and lower (29–62 %) than in Dietz et al. (2023a), while that of sequences recovered from dried samples was even lower (∼19 %). Alignment completeness of the sequences obtained from low coverage shotgun sequencing was highest (∼92 %), although the average coverage was much lower than for the target enrichment samples. We used the resulting phylogeny to reconstruct the historical biogeography of the group. To estimate a time-calibrated tree, we combined the mzl-USCO data of Pleophylla with a nucleotide alignment from an available transcriptomic dataset of Scarabaeoidea and used two different sets of secondary calibration points. Despite the problems associated with the capture rate of mzl-USCO sequences from museum specimens, we were able to infer a well-resolved phylogeny of the genus Pleophylla that also provided reliable estimates of the phylogenetic position of species for which we had little sequence data. Our study clearly identified South Africa as the geographic origin of Pleophylla. Timing and biogeographic history confirm a persistent fragmentation of forests since the Eocene. The occurrence of only one long-distance dispersal event from southern Africa to the Eastern African Arc even during the Miocene highlights the limited dispersal possibilities for these forest-adapted chafers, which do not seem to have had important northerly range expansions along hypothetical forest corridors during the Pleistocene.

Long-read sequencing and genome assembly of natural history collection samples and challenging specimens.

Museum collections harbor millions of samples, largely unutilized for long-read sequencing. Here, we use ethanol-preserved samples containing kilobase-sized DNA to show that amplification-free protocols can yield contiguous genome assemblies. Additionally, using a modified amplification-based protocol, employing an alternative polymerase to overcome PCR bias, we assembled the 3.1 Gb maned sloth genome, surpassing the previous 500 Mb protocol size limit. Our protocol also improves assemblies of other difficult-to-sequence molluscs and arthropods, including millimeter-sized organisms. By highlighting collections as valuable sample resources and facilitating genome assembly of tiny and challenging organisms, our study advances efforts to obtain reference genomes of all eukaryotes.

Systematic revision of the eastern Queensland land snail genus Figuladra Köhler and Bouchet, 2020 based on mitochondrial phylogenetics and comparative morpho-anatomy (Eupulmonata, Camaenidae)

ABSTRACT The current study revises the systematic taxonomy of Figuladra, a genus of camaenid land snails endemic to eastern Queensland, based on the investigation of many recently collected ethanol-preserved samples as well as historic museum material, including types. We employed comparative analyses to investigate the variation in morphological features (shell, penial anatomy) and performed phylogenetic analyses of mitochondrial 16S rDNA sequences to test species hypotheses. Phylogenetic analyses recovered three principal mitochondrial clades, one occurring in mid-east Queensland, one in south-east Queensland, and another in the upland areas of south-east Queensland. We recognise altogether 15 accepted species, including four that are newly described herein (i.e., Figuladra finlaysoni sp. nov., F. robertirwini sp. nov., F. bromileyorum sp. nov. and F. vidulus sp. nov.). The status of 11 previously described species has been assessed and revised diagnoses are provided for each of these. Species of the south-east Queensland clade revealed comparatively low levels of genetic and morphological differentiation and may represent examples of more recent, conceivably ongoing, speciation. Figuladra species have allopatric distributions with their ranges being separated by altitudinal or riverine barriers. Particularly the St Lawrence Gap dry corridor has been identified as a major biogeographic barrier separating two principal clades of Figuladra.

Application of palaeogenetic techniques to historic mollusc shells reveals phylogeographic structure in a New Zealand abalone.

Natural history collections worldwide contain a plethora of mollusc shells. Recent studies have detailed the sequencing of DNA extracted from shells up to thousands of years old and from various taphonomic and preservational contexts. However, previous approaches have largely addressed methodological rather than evolutionary research questions. Here, we report the generation of DNA sequence data from mollusc shells using such techniques, applied to Haliotis virginea Gmelin, 1791, a New Zealand abalone, in which morphological variation has led to the recognition of several forms and subspecies. We successfully recovered near-complete mitogenomes from 22 specimens including 12 dry-preserved shells up to 60 years old. We used a combination of palaeogenetic techniques that have not previously been applied to shell, including DNA extraction optimized for ultra-short fragments and hybridization-capture of single-stranded DNA libraries. Phylogenetic analyses revealed three major, well-supported clades comprising samples from: (1) The Three Kings Islands; (2) the Auckland, Chatham and Antipodes Islands; and (3) mainland New Zealand and Campbell Island. This phylogeographic structure does not correspond to the currently recognized forms. Critically, our nonreliance on freshly collected or ethanol-preserved samples enabled inclusion of topotypes of all recognized subspecies as well as additional difficult-to-sample populations. Broader application of these comparatively cost-effective and reliable methods to modern, historical, archaeological and palaeontological shell samples has the potential to revolutionize invertebrate genetic research.

A Feulgen glimpse into genome evolution during range expansion: a case study of the subterranean amphipod Niphargus schellenbergi

In 1923, Robert Feulgen designed a quantitative approach to measure the relative DNA content of a species (its so-called “C-value”, which is defined as half of the DNA content of a somatic cell and is expressed in pg). The Feulgen approach has several advantages: it can be used on ethanol-preserved samples, it is relatively inexpensive, and it does not require sophisticated equipment (Hardie et al. 2002, Jeffery and Gregory 2014, Kasten 2003). Gathering DNA content information is a key preliminary step for whole-genome sequencing of non-model species and also yield important information for ecological and evolutionary studies as well as for conservation (Jeffery et al. 2013), since genome size has been shown to be strongly correlated to extinction risk in animals and plants (Vinogradov 2004, Vinogradov 2003). Among invertebrates, Niphargidae is among the largest families of freshwater subterranean amphipods in the world, and more than 450 niphargid species have been described (Horton et al. 2021). Thus, it is considered an ideal model for evolutionary and ecological studies. Using a new, improved Feulgen protocol, we generated novel genome size estimates for 26 samples of Niphargus schellenbergi collected from central and western European niphargids. The obtained C-values ranged from 2.75 to 5.25 pg. Such a two-fold intraspecific variation is unusual but not unheard of: it occurs also in the monogonont rotifer Brachionus asplanchnoidis (Stelzer et al. 2021). Further research will be required to find out the mechanisms responsible for this variation, which may result from copy-number variation, polyploidy and/or various amounts of transposable elements.

Comparative evaluation of fish larval preservation methods on microbiome profiles to aid in metagenomics research.

Applications of microbiome research through metagenomics promise to generate microbiome manipulation strategies for improved larval survival in aquaculture. However, existing lacunae on the effects of sample preservation methods in metagenome profiles hinder the successful application of this technique. In this context, four preservation methods were scrutinized to identify reliable methods for fish larval microbiome research. The results showed that a total of ten metagenomics metrics, including DNA yield, taxonomic and functional microbiome profiles, and diversity measures, were significantly (P < 0.05) influenced by the preservation method. Activity ranking based on the performance and reproducibility showed that three methods, namely immediate direct freezing, room temperature preservation in absolute ethanol, and preservation at - 20°C in lysis, storage, and transportation buffer, could be recommended for larval microbiome research. Furthermore, as there was an apparent deviation of the microbiome profiles of ethanol preserved samples at room temperature, the other methods are preferred. Detailed analysis showed that this deviation was due to the bias towards Vibrionales and Rhodobacterales. The microbial taxa responsible for the dissimilarity across different methods were identified. Altogether, the paper sheds light on the preservation protocols of fish larval microbiome research for the first time. The results can help in cross-comparison of future and past larval microbiome studies. Furthermore, this is the first report on the activity ranking of preservation methods based on metagenomics metrics. Apart from methodological perspectives, the paper provides for the first time certain insights into larval microbial profiles of Rachycentron canadum, a potential marine aquaculture species. KEY POINTS: • First report on effects of preservation methods on fish larval microbiome profiles. • First report on activity ranking of preservation methods based on metagenomics metrics. • Storage methods influenced DNA yield, taxonomic and functional microbiome profiles.

Improved detection sensitivity using an optimal eDNA preservation and extraction workflow and its application to threatened sawfishes

Abstract Pressures on coastal ecosystems are increasing and aquatic species that are restricted to these habitats are facing the threat of extinction. However, the true extent of many threatened and rare aquatic species, especially elasmobranchs, remains unclear due to high levels of data deficiency and poor efficacy of traditional survey methods. Sawfishes (Pristidae), a family of shark‐like rays, are among the most threatened and rare elasmobranch species and are difficult to detect in turbid, coastal habitats. Reliable cost‐effective tools to detect these species are urgently needed to increase their conservation potential. Characterization of environmental DNA (eDNA) extracted from water samples has garnered significant appeal for detection of rare and threatened species. To assist conservation and monitoring efforts for sawfishes using eDNA, species‐specific TaqMan quantitative polymerase chain reaction assays were developed and validated to detect 1.25–5 copies of a 12S rRNA gene fragment. Filter samples were collected in Northern Territory, Australia to assess the utility of the developed eDNA assays and compare the efficacy of preservation and extraction workflows for detecting rare species. Dwarf sawfish (Pristis clavata) were detected in three of 20 sites, and there was a significant effect of preservation and extraction workflow on total eDNA yield and subsequent detection success. Longmire's preserved samples extracted using glycogen‐aided precipitation yielded a significantly higher concentration of total eDNA (n = 60; β = 1.27, t(95) = 8.172, P &lt; 0.0001) and yielded positive P. clavata eDNA detections compared to ethanol preserved samples extracted using QIAGEN DNeasy kit, which did not yield any positive detections. The optimized eDNA assays were developed to support monitoring efforts for endangered sawfishes. Importantly, this study demonstrates that choice of preservation and extraction workflow requires careful consideration, especially when detection of rare or threatened species can have important management and conservation outcomes.

A toolkit for studying Varroa genomics and transcriptomics: preservation, extraction, and sequencing library preparation

BackgroundThe honey bee parasite, Varroa destructor, is a leading cause of honey bee population declines. In addition to being an obligate ectoparasitic mite, Varroa carries several viruses that infect honey bees and act as the proximal cause of colony collapses. Nevertheless, until recently, studies of Varroa have been limited by the paucity of genomic tools. Lab- and field-based methods exploiting such methods are still nascent. This study developed a set of methods for preserving Varroa DNA and RNA from the field to the lab and processing them into sequencing libraries. We performed preservation experiments in which Varroa mites were immersed in TRIzol, RNAlater, and absolute ethanol for preservation periods up to 21 days post-treatment to assess DNA and RNA integrity.ResultsFor both DNA and RNA, mites preserved in TRIzol and RNAlater at room temperature degraded within 10 days post-treatment. Mites preserved in ethanol at room temperature and 4 °C remained intact through 21 days. Varroa mite DNA and RNA libraries were created and sequenced for ethanol preserved samples, 15 and 21 days post-treatment. All DNA sequences mapped to the V. destructor genome at above 95% on average, while RNA sequences mapped to V. destructor, but also sometimes to high levels of the deformed-wing virus and to various organisms.ConclusionsEthanolic preservation of field-collected mites is inexpensive and simple, and allows them to be shipped and processed successfully in the lab for a wide variety of sequencing applications. It appears to preserve RNA from both Varroa and at least some of the viruses it vectors.

The arachnological collections of the Museo Civico di Storia Naturale of Verona (Italy): an overview

The arachnological collections of the Museo Civico di Storia Naturale of Verona, Italy, contains roughly 20000 vials, including approximately 2000 species of Araneae and other Arachnida from all over the world. Within the collection, type material represents an important proportion with a count of more than 373 type series. All samples are distributed within four main collections: Brignoli, di Caporiacco, Museo Zangheri and‘Generale'. A further collection of 99% ethanol-preserved samples for molecular analysis is currently under preparation. A detailed list of all the arachnological type material stored in the museum is provided.

Microbiomes From Biorepositories? 16S rRNA Bacterial Amplicon Sequencing of Archived and Contemporary Intestinal Samples of Wild Mammals (Eulipotyphla: Soricidae)

Interest in gut microbial community composition has exploded recently as a result of the increasing ability to characterize these organisms and a growing understanding of their role in host fitness. New technologies, such as next generation amplicon (16S rRNA) sequencing, have enabled identification of bacterial communities from samples of diverse origin (e.g., fecal, skin, genital, environmental, etc.). Relatively little work, however, has explored the feasibility of utilizing historical samples (e.g., museum archived samples) of varying age, quality, and preservation type. Because natural history collections span multiple decades, these biorepositories have the potential to provide fundamental historical baselines to measure and better understand biodiversity on a changing planet. Utilizing even a small proportion of museum specimens could provide a means of sampling past microbial communities, allowing for direct comparison to contemporary communities and more complete understanding of dynamic shifts through time. We examined the feasibility of obtaining 16S rRNA amplicon microbiome data from whole gastrointestinal tracts (GIs) of shrews of varying age and preservation method, including 5 freshly collected shrew GIs immediately fixed in liquid nitrogen (LN2), 10 ten-year old shrew GIs frozen at -20°C (whole animal), and 10 shrews of varying ages (4 from 1968, 1 from 1980, 1 from 2001, 1 from 2004, 1 from 2007, 1 from 2011 and 2 from 2013) fixed and stored whole in 70% ethanol. Not surprisingly, results of 16S rDNA amplicon sequencing reveal significantly different bacterial communities between different preservation techniques and age of samples. Ten-year old frozen samples had bacterial communities most similar to freshly collected (LN2) samples, while the bacterial communities of both were significantly different from the 70% ethanol preserved samples of various ages. Amongst those preserved in 70% ethanol, age of samples also influenced bacterial community composition. Additionally, we compare results of OTU based and ASV based analyses. Looking ahead, field collectors and museums should develop and adopt best practices related to frozen preservation to ensure adequate material for future microbiome investigations.

Optimized and affordable high‐throughput sequencing workflow for preserved and nonpreserved small zooplankton specimens

Genomic analysis of hundreds of individuals is increasingly becoming standard in evolutionary and ecological research. Individual-based sequencing generates large amounts of valuable data from experimental and field studies, while using preserved samples is an invaluable resource for studying biodiversity in remote areas or across time. Yet, small-bodied individuals or specimens from collections are often of limited use for genomic analyses due to a lack of suitable extraction and library preparation protocols for preserved or small amounts of tissues. Currently, high-throughput sequencing in zooplankton is mostly restricted to clonal species, that can be maintained in live cultures to obtain sufficient amounts of tissue, or relies on a whole-genome amplification step that comes with several biases and high costs. Here, we present a workflow for high-throughput sequencing of single small individuals omitting the need for prior whole-genome amplification or live cultures. We establish and demonstrate this method using 27 species of the genus Daphnia, aquatic keystone organisms, and validate it with small-bodied ostracods. Our workflow is applicable to both live and preserved samples at low costs per sample. We first show that a silica-column based DNA extraction method resulted in the highest DNA yields for nonpreserved samples while a precipitation-based technique gave the highest yield for ethanol-preserved samples and provided the longest DNA fragments. We then successfully performed short-read whole genome sequencing from single Daphnia specimens and ostracods. Moreover, we assembled a draft reference genome from a single Daphnia individual (>50× coverage) highlighting the value of the workflow for non-model organisms.

Updated Morphological Descriptions of the Larval Stage of Urophycis (Family: Phycidae) from the Northeast United States Continental Shelf

Including early life history data in assessments can improve fisheries management by increasing our knowledge of stock structure, spawning habitat, and population trends. The identification of fish larvae to species is a necessary step in using early life history data toward this goal. Three species of hakes from the genus Urophycis are common on the northeast United States continental shelf: U. chuss or Red Hake, U. regia or Spotted Hake, and U. tenuis or White Hake. Unfortunately, identification of larval Urophycis has long been only possible at the genus level. Larvae of Urophycis (n = 277) collected in a subset of ethanol-preserved samples were identified genetically through sequencing of the cytochrome oxidase I gene and were used to update morphological descriptions with characters that separate these three species at the larval stage. Sequencing occurred in two stages: the first (n = 88) to develop a set of known-identity larvae to define species-specific traits, the second (n = 189) to test morphological identification based on the traits described in this study. We describe a combination of the location of dorsal and ventral pigment, head pigment, lower jaw pigment, and the timing of development of the pectoral fins to distinguish the larvae of these three species at sizes <6 mm. Using molecular techniques to improve morphological identifications is a powerful and efficient way to obtain the species-level data needed for assessments and management.

DNA Barcoding of Cyprinid Fish Chagunius chagunio Hamilton,1822 from Phewa Lake, Nepal

The present study is the first of its type that uses a technique of DNA barcoding to determine identification and relationship of a species of fish from Phewa lake, Nepal. The mitochondrial DNA from two ethanol-preserved samples of fish, randomly collected from Phewa lake, was extracted using Gene AllExgene TMtissue extraction kit. 650 base pair of mitochondrial cytochrome c oxidase subunit 1 (CO1) was amplified using a cocktail of four primers and was sequenced bidirectionaly using Sanger sequence method. The DNA sequences were edited using AliView software. The sequences confirmed Chagunius chagunio as their alignment with 16 reference sequences belonging to Chagunius chagunio in the NCBI GenBank, scored highest percentage of Query Cover (75% to 100%) and Percentage Identity (97.29% to 100%). The MEGA software analysed the DNA sequences to obtain their corresponding protein sequences. The DNA sequences were submitted to the GenBank and accession numbers (MN087472 and MN087473) were obtained. Clustal Omega software analysed multiple sequence alignment among 19 homologous DNA sequences of Chagunius chagunio from India, Bangladesh and Phewa lake, Nepal. The percentage of similarity among the aligned sequences was calculated as 39.3%. Based on the neighbour joining tree, the Chagunius chagunio of Phewa lake is found closely related with Chagunius chagunio of Bangladesh.

Variations in the microbiome due to storage preservatives are not large enough to obscure variations due to factors such as host population, host species, body site, and captivity

The study of the primate microbiome is critical in understanding the role of the microbial community in the host organism. To be able to isolate the main factors responsible for the differences observed in microbiomes within and between individuals, confounding factors due to technical variations need to be removed. To determine whether alterations due to preservatives outweigh differences due to factors such as host population, host species, body site, and habitat, we tested three methods (no preservative, 96% ethanol, and RNAlater) for preserving wild chimpanzee (fecal), wild lemur (fecal), wild vervet monkey (rectal, oral, nasal, otic, vaginal, and penile), and captive vervet monkey (rectal) samples. All samples were stored below - 20°C (short term) at the end of the field day and then at - 80°C until DNA extraction. Using 16S rRNA gene sequencing, we show a significant preservative effect on microbiota composition and diversity. Samples stored in ethanol and RNAlater appear to be less different compared with samples not stored in any preservative (none). Our differential analysis revealed significantly higher amounts of Enterococcaceae and Family XI in no preservative samples, Prevotellaceae and Spirochaetaceae in ethanol and RNAlater preserved samples, Oligosphaeraceae in ethanol-preserved samples, and Defluviitaleaceae in RNAlater preserved samples. While these preservative effects on the microbiome are not large enough to remove or outweigh the differences arising from biological factors (e.g., host species, body site, and habitat differences) they may promote misleading interpretations if they have large enough effect sizes compared to the biological factors (e.g., host population).

Preliminary analysis of New Zealand scampi (Metanephrops challengeri) diet using metabarcoding.

Deep sea lobsters are highly valued for seafood and provide the basis of important commercial fisheries in many parts of the world. Despite their economic significance, relatively little is known about their natural diets. Microscopic analyses of foregut content in some species have suffered from low taxonomic resolution, with many of the dietary items difficult to reliably identify as their tissue is easily digested. DNA metabarcoding has the potential to provide greater taxonomic resolution of the diet of the New Zealand scampi (Metanephrops challengeri) through the identification of gut contents, but a number of methodological concerns need to be overcome first to ensure optimum DNA metabarcoding results. In this study, a range of methodological parameters were tested to determine the optimum protocols for DNA metabarcoding, and provide a first view of M. challengeri diet. Several PCR protocols were tested, using two universal primer pairs targeting the 18S rRNA and COI genes, on DNA extracted from both frozen and ethanol preserved samples for both foregut and hindgut digesta. The selection of appropriate DNA polymerases, buffers and methods for reducing PCR inhibitors (including the use of BSA) were found to be critical. Amplification from frozen or ethanol preserved gut contents appeared similarly dependable. The COI gene was found to be more effective than 18S rRNA gene for identifying large eukaryotic taxa from the digesta; however, it was less successfully amplified. The 18S rRNA gene was more easily amplified, but identified mostly smaller marine organisms such as plankton and parasites. This preliminary analysis of the diet of M. challengeri identified a range of species (13,541 reads identified as diet), which included the ghost shark (Hydrolagus novaezealandiae), silver warehou (Seriolella punctata), tall sea pen (Funiculina quadrangularis) and the salp (Ihlea racovitzai), suggesting that they have a varied diet, with a high reliance on scavenging a diverse range of pelagic and benthic species from the seafloor.

Preservatives and sample preparation in stable isotope analysis of New Zealand freshwater invertebrates

ABSTRACTInterpretation of stable isotope data needs to take into account known, but often variable and inconsistent, influences of preservatives and lipids on carbon and nitrogen isotopic values. We tested how short-term (<6 months) preservation (ethanol and freezing) and preparation (lipid extraction) methods influence δ13C and δ15N values of 10 common New Zealand stream invertebrates and assessed whether mathematical lipid corrections (derived overseas) are valid here. The different methods significantly affected δ13C but not δ15N values. Specifically, ethanol-preserved and lipid-extracted samples were enriched in δ13C (by 1–2 ‰) relative to frozen samples. Short-term storage in ethanol (70%) was comparable to lipid removal and similar δ13C values were obtained when frozen samples were corrected mathematically. In contrast, δ15N values differed <0.1‰ across treatments. Together, this suggests that: (i) lipid extraction or mathematical correction may be unnecessary for ethanol-preserved samples, as ethanol appears to behave as a lipid solvent; (ii) existing mathematical corrections can adequately adjust for lipid-related shifts in δ13C in frozen samples; and (iii) δ15N values lend similar insight regardless of preservation/preparation methods. Although these results support the notion that archived (ethanol-preserved) invertebrate samples can inform isotope studies, additional work is needed to determine storage durations to which our findings apply.

Enzymatic detection of formalin-fixed museum specimens for DNA analysis and enzymatic maceration of formalin-fixed specimens

Abstract A simple enzymatic screening method has been developed to detect whether a tissue sample has been preserved with formalin or with ethanol only because such a method is a useful tool for predicting the quality of genetic test results. The method is based on enzymatic digestion at 55°C at neutral pH. The screening method shows that only ethanol-preserved tissue samples are dissolved, whereas formalin-preserved samples remain undissolved. The method was developed by the incorporation of laboratory rats preserved under controlled conditions in either 4% neutral buffered formalin or 96% ethanol. The method was subsequently tested on wild-living preserved specimens and an archived specimen. The protease enzyme used was Savinase® 16 L, Type EX from Novozymes A/S. The enzymatic screening test demands only simple laboratory equipment. The method is useful for natural history collections in museums where DNA analyses of archived specimens are performed. Wasted time and resources can be avoided through the detection of formalin-fixed specimens because these specimens yield low-quality, damaged DNA. In addition to the screening method, it is shown that formalin-preserved specimens can be macerated by enzymatic digestion under alkaline conditions at 55°C.

Mitochondrial Haplotype-Based Identification of Ethanol-Preserved Root-Knot Nematodes from Africa

The asexual root-knot nematodes (RKNs) (Meloidogyne spp.) exemplified by Meloidogyne incognita are widespread and damaging pests in tropical and subtropical regions worldwide. Comparison of amplification products of two adjacent polymorphic regions of the mitochondrial genome using DNA extracts of characterized RKN strains, including 15 different species, indicate that several species are derived from the same or closely related female lineages. Nevertheless, M. javanica, M. enterolobii, M. incognita, and other key species could each be assigned unique mitochondrial haplotypes based on polymerase chain reaction fragment size and restriction cleavage patterns. M. arenaria isolates did not group as a single haplotype, consistent with other reports of diversity within this species. To test the utility of this assay, we characterized ethanol-preserved samples from 103 single-species isolates from four countries in sub-Saharan Africa (Benin, Nigeria, Kenya, and Tanzania). Mitochondrial haplotypes corresponding to M. javanica and M. incognita were the most prevalent. Samples from western Africa included several instances of M. enterolobii but this species was not detected in samples from East Africa. This protocol provides progress toward a standardized strategy for identification of RKN species from small, preserved samples and a rational starting point for classifying species present in regions where previous knowledge has been limited.

DNA and Mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market

The morphological similarity among Sparidae species, which are characterized by a different market price, represents a serious problem for their trade and for stock management, since it encourages frauds for substitution. The most accredited morphological method for their identification is based on the dental-plate, but this approach is not simple and cannot be used for prepared products. When molecular methods are used the DNA degradation induced by cooking is the main drawback. In this work, we collected 314 reference tissues belonging to 75 Sparidae species and we produced a dataset of full (FDB) and mini-barcode (MDB) reference sequences starting from DNA extracted from fresh and ethanol-preserved tissues using universal primes. Moreover, some fresh samples were cooked. The FDB was successfully amplified in 91% (fresh), 50% (cooked) and 81% (ethanol-preserved) samples, while the amplification rates of the MDB were considerably higher in case of cooked (100%) and ethanol-preserved (94%) samples. The same primers were used for the amplification of the DNA obtained from 58 market samples (MS). All the DNA barcodes were compared with BOLD and GenBank using IDs and BLAST analysis. FDB was able to provide unambiguous species-level identifications for 53 (78%) and 44 (64.7%) reference samples analyzed on BOLD and GenBank, respectively. The Mini-DNA barcode (MDB) showed a lower discriminating power with 32 (45.7%) and 29 (41.4%) sequences unambiguously matched to a species on BOLD and GenBank. However, the MDB allowed to identify all the reference sequences as belonging to the Sparidae family. FDB and MDB showed a similar performance in analyzing the MS, allowing to highlight 21 (38%) mislabeled MS. Our study, while confirming the FDB as a reliable tool for fish authentication, proposes the MDB as a promising tool to recover molecular information in case of cooked products.

Mitochondrial and morphological differentiation in a previously unrecognized radiation of the land snail genus Parachloritis Ehrmann, 1912 on Timor (Pulmonata: Camaenidae)

The present work revises the taxonomy of one group of camaenid gastropods from Timor-Leste based on the study of a large number of recently collected ethanol preserved samples as well as historic museum material, including types. By employing comparative analyses of the variation in morphological features (shell, penial anatomy) and the differentiation in mitochondrial DNA sequences of the cytochrome c oxidase subunit 1 gene (COI) and the 16S rDNA (16S), altogether nineteen species are recognized from Timor-Leste and adjacent areas in the Lesser Sunda and Moluccas, such as West-Timor, Adonara, Leti and Sermata Islands (Indonesia). Four of these species were described previously and have mostly been placed within the genus Chloritis Beck, 1837 in the few historic treatments available. Fifteen species found to be new are formally described. In contrast to the previous taxonomic treatment, placement in the genus Parachloritis Ehrmann, 1912 is proposed on grounds of comparative shell morphology. Molecular phylogenetic analyses revealed that all examined species form a monophyletic group, which encompasses diverse shell forms. While most species have shells of the general chloritid type, which are of little taxonomic utility, highly distinct shell morphs (trochoid shells, dwarf forms) have originated within this radiation in independent lineages. Morphological change has occurred in some taxonomic lineages while the bulk of Parachloritis species has maintained an ancestral shell phenotype. This phenotypic stasis is attributed to stabilizing selection in species, which have maintained associations with ancestral habitats, while distinct shell forms have evolved as result of habitat shifts. Consequently, purely shellbased taxonomies are prone to errors due to misjudging the significance of shell characters. While some Parachloritis species uncovered here were found to be narrowly endemic, others had wide distributions that include more than one island. Narrow range endemism was predominantly found in dwarf species and in species that live in high altitudes.