Barcoding and traditional health practitioner perspectives are informative to monitor and conserve frogs and reptiles traded for traditional medicine in urban South Africa.

Previous literature suggests that Indigenous cultural practices, specifically traditional medicine, are commonplace among urban communities contrary to the general conception that such practices are restricted to rural societies. We reviewed previous literature for records of herptiles (frog and reptile species) sold by traditional health practitioners in urban South Africa, then used visual confirmation surveys, DNA barcoding and folk taxonomy to identify the herptile species that were on sale. Additionally, we interviewed 11 IsiZulu and SePedi speaking traditional health practitioners to document details of the collection and pricing of herptile specimens along with the practitioners' views of current conservation measures for traditional medicine markets. The 34 herptile species recorded in previous literature on traditional medicine markets included endangered and non-native species. Spectrophotometry measurements of the DNA we extracted from the tissue of herptiles used in traditional medicine were an unreliable predictor of whether those extractions would be suitable for further experimental work. From our initial set of 111 tissue samples, 81 sequencing reactions were successful and 55 of those sequences had species-level matches to COI reference sequences on the NCBI GenBank and/or BOLD databases. Molecular identification revealed that traditional health practitioners correctly labelled 77% of the samples that we successfully identified with DNA barcoding in this study. Our mixed methodology approach is useful for conservation planning as it updates knowledge of animal use in Indigenous remedies and can accurately identify species of high conservation priority. Furthermore, this study highlights the possibility of collaborative conservation planning with traditional health practitioners.

Rediscovery, range extension, phylogenetic relationships and updated diagnosis of the Ornate Long-tailed Lizard Latastia ornata Monard, 1940 (Squamata: Lacertidae).

The lacertid Latastia ornata was known to date only by its holotype collected in 1938 in Bafatá, central Guinea-Bissau. We report new specimens and localities from Guinea-Conakry, a new country record and major range extension of 700 km SE of the type-locality. We provide an updated diagnosis of the species, including the first genetic and osteological data, and confirm that Latastia ornata is closely related to, but distinct from, L. longicaudata based on external morphology, cranial osteology, DNA data and zoogeography.

Cypress terpenes in sawfly larva of Susana cupressi (Hymenoptera: Symphyta: Tenthredinoidea).

Several sawfly species (Hymenoptera: Symphyta) possess larval stages with oesophageal diverticula in which plant compounds are sequestered and used for defence against predators. These organs are present in the larvae of Susana (Tenthredinidae) but remain poorly studied. Here, the aim was to analyse the diverticula extract of Susana cupressi by gas chromatography-mass spectrometry to better understand the ecology of this species. The foliage of the hostplant (Cupressus sempervirens), as well as the larval foregut, midgut, and haemolymph were also analysed. Complementary data were gathered by morphological observations, bioassays using ants, and genetic analyses to identify the studied Susana species. Altogether, 48 terpenes were identified, 30 being sesquiterpenes. The terpenes were generally detected in the foliage, but also in the diverticula, foregut, and midgut, whereas none of them in the haemolymph. The main compounds were alpha-cedrene, alpha-fenchene, alpha-pinene, alpha-terpinyl acetate, beta-myrcene, beta-pinene, cedrol, delta 3-carene, epi-bicyclosesquiphellandrene, germacrene D, limonene, sabinene, and terpinolene. The chemical profiles of these 13 compounds were significantly correlated between foliage-diverticula, diverticula-foregut and foregut-midgut, but not correlated for the three remaining possible comparisons. Alpha-pinene decreased and germacrene D increased from the foliage to the diverticula, which may reflect a specific sequestration of the latter terpene and its known deleterious effects on insects. We conclude that larvae of S. cupressi, similarly to those of diprionids, are well defended against predatory attacks by sequestering and regurgitating hostplant terpenes, including germacrene D.

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

DNA barcoding echinoderms from the East Coast of South Africa. The challenge to maintain DNA data connected with taxonomy.

Echinoderms are marine water invertebrates that are represented by more than 7000 extant species, grouped in five classes and showing diverse morphologies (starfish, sea lilies, feather stars, sea urchins, sea cucumbers, brittle and basket stars). In an effort to further study their diversity, DNA barcodes (DNA fragments of the 5' end of the cytochrome c oxidase subunit I gene, COI) have been used to complement morphological examination in identifying evolutionary lineages. Although divergent clusters of COI sequences were reported to generally match morphological species delineations, they also revealed some discrepancies, suggesting overlooked species, ecophenotypic variation or multiple COI lineages within one species. Here, we sequenced COI fragments of 312 shallow-water echinoderms of the East Coast of South Africa (KwaZulu-Natal Province) and compared morphological identifications with species delimitations obtained with four methods that are exclusively based on COI sequences. We identified a total of 103 morphospecies including 18 that did not exactly match described species. We also report 46 COI sequences that showed large divergences (>5% p-distances) with those available to date and publish the first COI sequences for 30 species. Our analyses also identified discordances between morphological identifications and COI-based species delimitations for a considerable proportion of the morphospecies studied here (49/103). For most of them, further investigation is necessary to keep a sound connection between taxonomy and the growing importance of DNA-based research.

Population genomics of introduced Nile tilapia Oreochromis niloticus (Linnaeus, 1758) in the Democratic Republic of the Congo: Repeated introductions since colonial times with multiple sources.

During colonial times, Nile tilapia Oreochromis niloticus (Linnaeus, 1758) was introduced into non-native parts of the Congo Basin (Democratic Republic of the Congo, DRC) for the first time. Currently, it is the most farmed cichlid in the DRC, and is present throughout the Congo Basin. Although Nile tilapia has been reported as an invasive species, documentation of historical introductions into this basin and its consequences are scant. Here, we study the genetic consequences of these introductions by genotyping 213 Nile tilapia from native and introduced regions, focusing on the Congo Basin. Additionally, 48 specimens from 16 other tilapia species were included to test for hybridization. Using RAD sequencing (27,611 single nucleotide polymorphisms), we discovered genetic admixture with other tilapia species in several morphologically identified Nile tilapia from the Congo Basin, reflecting their ability to interbreed and the potential threat they pose to the genetic integrity of native tilapias. Nile tilapia populations from the Upper Congo and those from the Middle-Lower Congo are strongly differentiated. The former show genetic similarity to Nile tilapia from the White Nile, while specimens from the Benue Basin and Lake Kariba are similar to Nile tilapia from the Middle-Lower Congo, suggesting independent introductions using different sources. We conclude that the presence of Nile tilapia in the Congo Basin results from independent introductions, reflecting the dynamic aquaculture history, and that their introduction probably leads to genetic interactions with native tilapias, which could lower their fitness. We therefore urge avoiding further introductions of Nile tilapia in non-native regions and to use native tilapias in future aquaculture efforts.

A masculinizing supergene underlies an exaggerated male reproductive morph in a spider.

In many species, individuals can develop into strikingly different morphs, which are determined by a simple Mendelian locus. How selection shapes loci that control complex phenotypic differences remains poorly understood. In the spider Oedothorax gibbosus, males develop either into a 'hunched' morph with conspicuous head structures or as a fast-developing 'flat' morph with a female-like appearance. We show that the hunched-determining allele contains a unique genomic fragment of approximately 3 megabases that is absent in the flat-determining allele. This fragment comprises dozens of genes that duplicated from genes found at the same as well as different chromosomes. All functional duplicates, including a duplicate of the key sexual differentiation regulatory gene doublesex, show male-specific expression, which illustrates their integrated role as a masculinizing supergene. Our findings demonstrate how extensive indel polymorphisms and duplications of regulatory genes may contribute to the evolution of co-adapted gene clusters, sex-limited reproductive morphs and the enigmatic evolution of exaggerated sexual traits in general.

Exploring the use of micro-computed tomography (micro-CT) in the taxonomy of sea cucumbers: a case-study on the gravel sea cucumber Neopentadactylamixta (Östergren, 1898) (Echinodermata, Holothuroidea, Phyllophoridae).

Sea cucumber taxonomy and systematics has in the past heavily relied on gross external and internal anatomy, ossicle assemblage in different tissues, and molecular characterisation, with coloration, habitat, and geographical and bathymethric distribution also considered important parameters. In the present paper, we made these observations and techniques in detail and complemented them with the novel technique of micro-computed tomography of the calcareous ring. We investigated a single European species, the so-called gravel sea cucumber, Neopentadactylamixta (Östergren, 1898), using recently collected material from the Chausey Islands, Normandy, France. We redescribed the species, illustrated its ossicle assemblage through scanning electron microscopy, and visualised the calcareous ring through stacking photography and through micro-CT scanning. Additionally, a DNA fragment of 955 base pairs of the 18S ribosomal RNA gene was sequenced from one specimen, which showed a high similarity with the only sequence of N.mixta publicly available. We completed this integrative study by providing a detailed distribution of the occurrence of N.mixta based on published, verifiable accounts.

Cryptophyllium, the hidden leaf insects - descriptions of a new leaf insect genus and thirteen species from the former celebicum species group (Phasmatodea, Phylliidae).

While the leaf insects (Phylliidae) are a well-supported group within Phasmatodea, the genus Phyllium Illiger, 1798 has repeatedly been recovered as paraphyletic. Here, the Phyllium (Phyllium) celebicum species group is reviewed and its distinctiveness from the remaining Phylliini genera and subgenera in a phylogenetic context based on morphological review and a phylogenetic analysis of three genes (nuclear gene 28S and mitochondrial genes COI and 16S) from most known and multiple undescribed species is shown. A new genus, Cryptophyllium gen. nov., is erected to partially accommodate the former members of the celebicum species group. Two species, Phyllium ericoriaiHennemann et al., 2009 and Phyllium bonifacioi Lit & Eusebio, 2014 morphologically and molecularly do not fall within this clade and are therefore left within Phyllium (Phyllium). The transfer of the remaining celebicum group members from Phyllium Illiger, 1798 to this new genus creates the following new combinations; Cryptophyllium athanysus (Westwood, 1859), comb. nov.; Cryptophyllium celebicum (de Haan, 1842), comb. nov.; Cryptophyllium chrisangi (Seow-Choen, 2017), comb. nov.; Cryptophyllium drunganum (Yang, 1995), comb. nov.; Cryptophyllium oyae (Cumming & Le Tirant, 2020), comb. nov.; Cryptophyllium parum (Liu, 1993), comb. nov.; Cryptophyllium rarum (Liu, 1993), comb. nov.; Cryptophyllium tibetense (Liu, 1993), comb. nov.; Cryptophyllium westwoodii (Wood-Mason, 1875), comb. nov.; Cryptophyllium yapicum (Cumming & Teemsma, 2018), comb. nov.; and Cryptophyllium yunnanense (Liu, 1993), comb. nov. The review of specimens belonging to this clade also revealed 13 undescribed species, which are described within as: Cryptophyllium animatum gen. et sp. nov. from Vietnam: Quang Nam Province; Cryptophyllium bankoi gen. et sp. nov. from Vietnam: Quang Ngai, Thua Thien Hue, Da Nang, Gia Lai, Quang Nam, and Dak Nong Provinces; Cryptophyllium bollensi gen. et sp. nov. from Vietnam: Ninh Thuan Province; Cryptophyllium daparo gen. et sp. nov. from China: Yunnan Province; Cryptophyllium echidna gen. et sp. nov. from Indonesia: Wangi-wangi Island; Cryptophyllium faulkneri gen. et sp. nov. from Vietnam: Quang Ngai and Lam Dong Provinces; Cryptophyllium icarus gen. et sp. nov. from Vietnam: Lam Dong and Dak Lak Provinces; Cryptophyllium khmer gen. et sp. nov. from Cambodia: Koh Kong and Siem Reap Provinces; Cryptophyllium limogesi gen. et sp. nov. from Vietnam: Lam Dong, Dak Lak, and Dak Nong Provinces; Cryptophyllium liyananae gen. et sp. nov. from China: Guangxi Province; Cryptophyllium nuichuaense gen. et sp. nov. from Vietnam: Ninh Thuan Province; Cryptophyllium phami gen. et sp. nov. from Vietnam: Dong Nai and Ninh Thuan Provinces; and Cryptophyllium wennae gen. et sp. nov. from China: Yunnan Province. All newly described species are morphologically described, illustrated, and molecularly compared to congenerics. With the molecular results revealing cryptic taxa, it was found necessary for Cryptophyllium westwoodii (Wood-Mason, 1875), comb. nov. to have a neotype specimen designated to allow accurate differentiation from congenerics. To conclude, male and female dichotomous keys to species for the Cryptophyllium gen. nov. are presented.

Mitogenomic characterization and systematic placement of the Congo blind barb Caecobarbus geertsii (Cypriniformes: Cyprinidae).

This study presents the first complete mitochondrial genome (mitogenome) of Caecobarbus geertsii, the Congo blind barb, a cave-dwelling, CITES-protected, cyprinid fish endemic to the Lower Congo basin (DRC). The length of the circular mitogenome is 16,565 base pairs. The 13 protein coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes are similar in position and direction to those of other members of the family Cyprinidae. Phylogenetic analyses including 28 complete mitogenomes from representatives of the subfamily Smiliogastrinae (Cyprinidae), showed that Caecobarbus was nested within a clade including representatives of the genus Enteromius. The data presented in this study provide information on the molecular identification and classification of this threatened species. The results further suggest the need for a taxonomic revision of the genus Enteromius.

Ancient DNA suggests modern wolves trace their origin to a Late Pleistocene expansion from Beringia.

Grey wolves (Canis lupus) are one of the few large terrestrial carnivores that have maintained a wide geographical distribution across the Northern Hemisphere throughout the Pleistocene and Holocene. Recent genetic studies have suggested that, despite this continuous presence, major demographic changes occurred in wolf populations between the Late Pleistocene and early Holocene, and that extant wolves trace their ancestry to a single Late Pleistocene population. Both the geographical origin of this ancestral population and how it became widespread remain unknown. Here, we used a spatially and temporally explicit modelling framework to analyse a data set of 90 modern and 45 ancient mitochondrial wolf genomes from across the Northern Hemisphere, spanning the last 50,000 years. Our results suggest that contemporary wolf populations trace their ancestry to an expansion from Beringia at the end of the Last Glacial Maximum, and that this process was most likely driven by Late Pleistocene ecological fluctuations that occurred across the Northern Hemisphere. This study provides direct ancient genetic evidence that long-range migration has played an important role in the population history of a large carnivore, and provides insight into how wolves survived the wave of megafaunal extinctions at the end of the last glaciation. Moreover, because Late Pleistocene grey wolves were the likely source from which all modern dogs trace their origins, the demographic history described in this study has fundamental implications for understanding the geographical origin of the dog.

First mitochondrial genomes of five hoverfly species of the genus Eristalinus (Diptera: Syrphidae).

The hoverfly genus Eristalinus (Diptera, Syrphidae) contains many widespread pollinators. The majority of the species of Eristalinus occur in the Afrotropics and their molecular systematics still needs to be investigated. This study presents the first complete and annotated mitochondrial genomes for five species of Eristalinus. They were obtained by high-throughput sequencing of total genomic DNA. The total length of the mitogenomes varied between 15 757 and 16 245 base pairs. Gene composition, positions, and orientation were shared across species, and were identical to those observed for other Diptera. Phylogenetic analyses (maximum likelihood and Bayesian inference) based on the 13 protein coding and both rRNA genes suggested that the subgenus Eristalinus was paraphyletic with respect to the subgenus Eristalodes. An analysis of the phylogenetic informativeness of all protein coding and rRNA genes suggested that NADH dehydrogenase subunit 5 (nad5), cytochrome c oxidase subunit 1, nad4, nad2, cytochrome b, and 16S rRNA genes are the most promising mitochondrial molecular markers to result in supported phylogenetic hypotheses of the genus. In addition to the five complete mitogenomes currently available for hoverflies, the five mitogenomes published here will be useful for broader molecular phylogenetic analyses among hoverflies.

DNA barcoding fishes from the Congo and the Lower Guinean provinces: Assembling a reference library for poorly inventoried fauna.

The Congolese and Lower Guinean ichthyological provinces are understudied hotspots of the global fish diversity. Here, we barcoded 741 specimens from the Lower and Middle Congo River and from three major drainage basins of the Lower Guinean ichthyological province, Kouilou-Niari, Nyanga and Ogowe. We identified 194 morphospecies belonging to 82 genera and 25 families. Most morphospecies (92.8%) corresponded to distinct clusters of DNA barcodes. Of the four morphospecies present in both neighbouring ichthyological provinces, only one showed DNA barcode divergence <2.5%. A small fraction of the fishes barcoded here (12.9% of the morphospecies and 16.1% of the barcode clusters representing putative species) were also barcoded in a previous large-scale DNA analysis of freshwater fishes of the Lower Congo published in 2011 (191 specimens, 102 morphospecies). We compared species assignments before and after taxonomic updates and across studies performed by independent research teams and observed that most cases of inconsistent species assignments were due to unknown diversity (undescribed species and unknown intraspecific variation). Our results report more than 17 putative new species and show that DNA barcode data provide a measure of genetic variability that facilitates the inventory of underexplored ichthyofaunae. However, taxonomic scrutiny, associated with revisions and new species descriptions, is indispensable to delimit species and build a coherent reference library.

Significant loss of mitochondrial diversity within the last century due to extinction of peripheral populations in eastern gorillas.

Species and populations are disappearing at an alarming rate as a direct result of human activities. Loss of genetic diversity associated with population decline directly impacts species' long-term survival. Therefore, preserving genetic diversity is of considerable conservation importance. However, to assist in conservation efforts, it is important to understand how genetic diversity is spatially distributed and how it changes due to anthropogenic pressures. In this study, we use historical museum and modern faecal samples of two critically endangered eastern gorilla taxa, Grauer's (Gorilla beringei graueri) and mountain gorillas (Gorilla beringei beringei), to directly infer temporal changes in genetic diversity within the last century. Using over 100 complete mitochondrial genomes, we observe a significant decline in haplotype and nucleotide diversity in Grauer's gorillas. By including historical samples from now extinct populations we show that this decline can be attributed to the loss of peripheral populations rather than a decrease in genetic diversity within the core range of the species. By directly quantifying genetic changes in the recent past, our study shows that human activities have severely impacted eastern gorilla genetic diversity within only four to five generations. This rapid loss calls for dedicated conservation actions, which should include preservation of the remaining peripheral populations.

Species limits, interspecific hybridization and phylogeny in the cryptic land snail complex Pyramidula: The power of RADseq data.

Restriction site-associated DNA sequencing (RADseq) was used to jointly assess phylogenetic relationships, interspecific hybridization and species delimitation in the cryptic, non-model land snail complex Pyramidula. A robust phylogeny was inferred using a matrix of concatenated sequences of almost 1,500,000bp long, containing >97,000 polymorphic sites. Maximum likelihood analyses fully resolved the phylogenetic relationships among species and drastically improved phylogenetic trees obtained from mtDNA and nDNA gene trees (COI, 16S rRNA, 5.8S rRNA, ITS2 and 28S rRNA sequence data). The best species delimitation scenario was selected on the basis of 875 unlinked single nucleotide polymorphisms, showing that nine Pyramidula species should be distinguished in Europe. Applying D-statistics provided no or weak evidence of interspecific hybridization among Pyramidula, except for some evidence of gene flow between two species.

DNA barcoding and male genital morphology reveal five new cryptic species in the West Palearctic bee Seladonia smaragdula (Vachal, 1895) (Hymenoptera: Apoidea: Halictidae).

Several forms or variants have long been recognized in the West Palearctic sweat bee Seladonia smaragdula (Vachal, 1895). Using DNA barcoding and morphological characters, primarily of the male genitalia, these variants are here recognized and described as five new species: S. gemmella Pauly sp. nov., S. submediterranea Pauly sp. nov., S. orientana Pauly & Devalez sp. nov., S. phryganica Pauly & Devalez sp. nov., and S. cretella Pauly & Devalez sp. nov. Also, we designate a lectotype for Halictus smaragdulus Vachal, consider Seladonia butea (Warncke, 1975) and S. morinella (Warncke, 1975) as nomina dubia, and discuss the identity of the Seladonia specimens from Australia currently determined as S. smaragdula.

Taxonomy, phylogeny and host plants of some Abia sawflies (Hymenoptera, Cimbicidae).

We briefly review the taxonomy of Abia, and attempt to clarify their systematics by phylogenetic tree reconstructions inferred from three (nuclear and mitochondrial) genes of some West Palaearctic and Nearctic species. The main question which we asked is whether the distinction, made by several authors, of two genera within this group is justified. Based on the species here sampled, our results strongly support a clade recognised widely in earlier literature as Abia or Abia (Abia), but do not always support another clade, Zaraea or Abia (Zaraea), as monophyletic. In the interests of nomenclatural stability and for other practical reasons, the two nominal genera should be treated as synonyms. Host plant associations may be useful in the systematics of Abia species, but this topic requires further investigation and inclusion of more species in phylogenetic analyses.

Genetic and morphological divergences in the cosmopolitan deep-sea amphipod Eurythenes gryllus reveal a diverse abyss and a bipolar species.

Eurythenes gryllus is one of the most widespread amphipod species, occurring in every ocean with a depth range covering the bathyal, abyssal and hadal zones. Previous studies, however, indicated the existence of several genetically and morphologically divergent lineages, questioning the assumption of its cosmopolitan and eurybathic distribution. For the first time, its genetic diversity was explored at the global scale (Arctic, Atlantic, Pacific and Southern oceans) by analyzing nuclear (28S rDNA) and mitochondrial (COI, 16S rDNA) sequence data using various species delimitation methods in a phylogeographic context. Nine putative species-level clades were identified within E. gryllus. A clear distinction was observed between samples collected at bathyal versus abyssal depths, with a genetic break occurring around 3,000 m. Two bathyal and two abyssal lineages showed a widespread distribution, while five other abyssal lineages each seemed to be restricted to a single ocean basin. The observed higher diversity in the abyss compared to the bathyal zone stands in contrast to the depth-differentiation hypothesis. Our results indicate that, despite the more uniform environment of the abyss and its presumed lack of obvious isolating barriers, abyssal populations might be more likely to show population differentiation and undergo speciation events than previously assumed. Potential factors influencing species' origins and distributions, such as hydrostatic pressure, are discussed. In addition, morphological findings coincided with the molecular clades. Of all specimens available for examination, those of the bipolar bathyal clade seemed the most similar to the 'true' E. gryllus. We present the first molecular evidence for a bipolar distribution in a macro-benthic deep-sea organism.

Taxonomic and population genetic re-interpretation of two color morphs of the decollate snail, Rumina decollata (Mollusca, Pulmonata) in southern France.

The hermaphroditic terrestrial snail Rumina decollata has a mixed breeding system with a high prevalence of self-fertilization. In the Montpellier area (France), the species is represented by a dark and a light color morph. Based on allozyme data, both morphs have been reported as single, homozygous multilocus genotypes (MLG), differing at 13 out of 26 loci, but still showing occasional hybridization. Recent DNA sequence data suggest that each morph is a different phylogenetic species. In order to further evaluate this new taxonomic interpretation, the present contribution explores to what extent populations or color morphs indeed consist of single or few MLG. As such it is shown that both morphs are not single, homozygous MLG, but instead reveal a considerable amount of allelic variation and substantial numbers of heterozygous microsatellite genotypes. This suggests that outcrossing may be more prevalent than previously reported. Nevertheless, both morphs maintain a diagnostic multimarker differentiation in the presence of outcrossing in sympatric conditions, implying that they may be interpreted as species under the biological species concept. Finally, our data challenge the idea that simultaneous hermaphrodites should be either strict selfers or strict outcrossers.

Exploring species level taxonomy and species delimitation methods in the facultatively self-fertilizing land snail genus Rumina (gastropoda: pulmonata).

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.