Matthias F. Geiger

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule‐coalescent (GMYC) model‐based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA‐barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large‐scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.

Molecular diversity of Germany's freshwater fishes and lampreys assessed by DNA barcoding

This study represents the first comprehensive molecular assessment of freshwater fishes and lampreys from Germany. We analysed COI sequences for almost 80% of the species mentioned in the current German Red List. In total, 1056 DNA barcodes belonging to 92 species from all major drainages were used to (i) build a reliable DNA barcode reference library, (ii) test for phylogeographic patterns, (iii) check for the presence of barcode gaps between species and (iv) evaluate the performance of the barcode index number (BIN) system, available on the Barcode of Life Data Systems. For over 78% of all analysed species, DNA barcodes are a reliable means for identification, indicated by the presence of barcode gaps. An overlap between intra‐ and interspecific genetic distances was present in 19 species, six of which belong to the genus Coregonus. The Neighbour‐Joining phenogram showed 60 nonoverlapping species clusters and three singleton species, which were related to 63 separate BIN numbers. Furthermore, Barbatula barbatula, Leucaspius delineatus, Phoxinus phoxinus and Squalius cephalus exhibited remarkable levels of cryptic diversity. In contrast, 11 clusters showed haplotype sharing, or low levels of divergence between species, hindering reliable identification. The analysis of our barcode library together with public data resulted in 89 BINs, of which 56% showed taxonomic conflicts. Most of these conflicts were caused by the use of synonymies, inadequate taxonomy or misidentifications. Moreover, our study increased the number of potential alien species in Germany from 14 to 21 and is therefore a valuable groundwork for further faunistic investigations.

Cobitis avicennae, a new species of spined loach from the Tigris River drainage (Teleostei: Cobitidae).

Cobitis avicennae, new species, from the Karkheh and Karun sub-drainages in the Tigris catchment is distinguished from other Cobitis species in the Persian Gulf, Kor and the southern Caspian Sea basins by having a single lamina circularis in males, a small comma-shaped black spot on the upper caudal-fin base, 5½ branched anal-fin rays, 5-6 rows of dark spots on the dorsal and caudal fins, scales below the dorsal-fin base with a small focal zone and pigmentation zone Z4 with 12-17 large, partly fused blotches. It is also distinguished from other Cobitis species in the comparison group by six fixed, diagnostic nucleotide substitutions in the mtDNA COI barcode region.

Combining geometric morphometrics with molecular genetics to investigate a putative hybrid complex: a case study with barbels Barbus spp. (Teleostei: Cyprinidae)

This integrative study examined the morphological and genetic affinities of three endemic barbel species from Italy (brook barbel Barbus caninus, Italian barbel Barbus plebejus and horse barbel Barbus tyberinus) and of putative hybrid specimens to their species of origin. Two of the species frequently occur together with the non-native barbel Barbus barbus. DNA barcoding indicates that mitochondrial (mt) haplotypes often do not match the species expected from morphology. Linear distance measurements and meristics are not informative for discrimination of the species and putative hybrids, but a discriminant analysis of principal components (DAPC) of geometric landmark data produces reassignments largely in congruence with mt and nuclear genetic data. Cyto-nuclear conflicts confirm the presence of hybridization in B. plebejus and B. tyberinus and identify additional introgressed specimens. A comparison between mixed genotypes and their morphology-based assignment reveals no predictable pattern. The finding that most individuals of the morphologically similar B. plebejus and B. tyberinus have very high assignment probabilities to their respective species suggests that the presented approach may serve as a valuable tool to distinguish morphologically very similar taxa.

Squalius namak , a new chub from Lake Namak basin in Iran (Teleostei: Cyprinidae)

Squalius namak, new species, from the endorheic Lake Namak and Kavir basins in Iran, is distinguished from the species of the genus Squalius in the Persian Gulf and the southern Caspian Sea basins by having a wide and thick symphysial knob on the lower jaw, a convex posterior anal-fin margin, a bold, dark-grey or brown, roundish or crescent-shaped blotch at the posterior tip of each flank scale and orange caudal-, anal- and pelvic-fin rays in life. Squalius namak is also characterized by four fixed, diagnostic nucleotide substitutions in the mtDNA COI barcode region.

Aphanius marassantensis , a new toothcarp from the Kızılırmak drainage in northern Anatolia (Cyprinodontiformes: Cyprinodontidae)

Aphanius marassantensis, new species, is described from the Kızılırmak River drainage in northern Anatolia based on colouration, meristic and morphometric characters, and the mtDNA COI barcode region. It is distinguished from other Anatolian Aphanius by one or several of the following characters: a stout body shape (BD/SL 28.2-39.6%), complete scale cover, and 25-28 scales along the lateral line. Males have 8-13 dark-brown lateral bars, of which the antepenultimate bar anterior to the caudal-fin base is 0.9-1.8 times wider than the anterior white interspace, 2-3 vertical rows of spots on the caudal fin, a black dorsal fin, sometimes with a narrow whitish-grey base, a white anal fin with 1-3 rows of black spots, in some individuals with a black margin, and hyaline pelvic fins. Females do not have vertical rows of dark-brown spots on caudal or anal fins, but numerous dark-brown spots on the flanks, arranged in 1-3 lateral rows behind a vertical from the dorsal-fin base. Their dorsal fin is hyaline with tiny dark-brown spots on rays and membranes; pectoral fins, caudal and anal fins are hyaline, and one prominent large dark-brown blotch is situated in mid-lateral position on the hypural plate. The new species is also distinguished by 11 fixed, diagnostic nucleotide substitutions in the mtDNA COI barcode region. The description of this new species, which brings the number of Anatolian Aphanius species to 12, underlines the character of Anatolia as a region of extraordinarily high biodiversity.

Sasanidus , a new generic name for Noemacheilus kermanshahensis Bănărescu & Nalbant, with discussion of Ilamnemacheilus and Schistura (Teleostei; Nemacheilidae)

Sasanidus, new genus, is described for Noemacheilus kermanshahensis Bănărescu & Nalbant, endemic to the Karkheh and Karun drainages in Iran. Sasanidus kermanshahensis was initially identified as a species in Oxynoemacheilus, from which it is distinguished by the absence of an external sexual dimorphism (i.e. longer pectoral fin, and nuptial tubercles on fins, head and back in males). Sasanidus is distinguished from all other genera of Nemacheilidae in the Middle East by a combination of the following character states: pelvic-fin origin behind of a vertical of the dorsal-fin origin, anus about one eye diameter in front of the anal-fin origin, dorsal adipose keel absent, a high crest on the bony capsule of the swim bladder present and colour pattern marbled or mottled or with an irregularly shaped midlateral stripe. Ilamnemacheilus longipinnis was examined and no difference could be found between Ilamnemacheilus and Oxynoemacheilus. Therefore, Ilamnemacheilus is treated as a synonym of Oxynoemacheilus. COI barcode sequences from all nemacheilid loach genera occurring in the Middle East and western India are analysed jointly for the first time. The view that Schistura is a paraphyletic assemblage is supported by the clustering of DNA sequences from 45 specimens placed in at least 20 species in the genus Schistura analysed here.

Eidinemacheilus proudlovei , a new subterranean loach from Iraqi Kurdistan (Teleostei; Nemacheilidae)

Eidinemacheilus proudlovei, new species, is described from subterranean waters in the Little Zab River drainage in Iraqi Kurdistan. After the discovery of E. smithi in 1976, E. proudlovei is the second troglomorphic nemacheilid loach found in the Middle East and the second species placed in Eidinemacheilus. Eidinemacheilus proudlovei is distinguished from E. smithi by having 8+8 or 8+7 branched caudal-fin rays, no adipose keel on the caudal peduncle, enlarged jaws and a fully developed head canal system. It furthers differs substantially in its DNA barcode (>8% K2P distance) from all other nemacheilid loaches in the Middle East, Europe and Western India.

DNA metabarcoding from sample fixative as a quick and voucher preserving biodiversity assessment method

Metabarcoding is a powerful tool for biodiversity assessment and has become increasingly popular in recent years. Although its reliability and applicability have been proven in numerous scientific studies, metabarcoding still suffers from some drawbacks. One is the usually mandatory destruction of specimens before DNA extraction, which is problematic because it does not allow a later taxonomic evaluation of the results. Additionally, metabarcoding often implements a time-consuming step, where specimens need to be separated from substrate or sorted in different size classes. A non-destructive protocol, excluding any sorting step, where the extraction of DNA is conducted from a samples fixative (ethanol) could serve as an alternative. We test an innovative protocol, where the sample preserving ethanol is filtered and DNA extracted from the filter for subsequent DNA metabarcoding. We first tested the general functionality of this approach on 15 mock communities comprising one individual of eight different macroinvertebrate taxa each and tried to increase DNA yield through different treatments (ultrasonic irradiation, shaking, freezing). Application of the method was successful for most of the samples and taxa, but showed weaknesses in detecting mollusc taxa. In a second step, the community composition detected in DNA from ethanol was compared to conventional bulk sample metabarcoding of complex environmental samples. We found that especially taxa with pronounced exoskeleton or shells (Coleoptera, Isopoda) and small taxa (Trombidiformes) were underrepresented in ethanol samples regarding taxa diversity and read numbers. However, read numbers of Diptera (mainly chironomids) and Haplotaxida were higher in ethanol derived DNA samples, which might indicate the detection of stomach content, which would be an additional advantage of this approach. Concerning EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa which are decisive for the determination of ecological statuses, both methods had 46 OTUs in common with 4 unique to the ethanol samples and 10 to the bulk samples. Results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. For a comprehensive picture on total biodiversity, the method might however not be sufficient and conventional bulk sample metabarcoding should be applied.

Aphanius kruppi , a new killifish from Oman with comments on the A. dispar species group (Cyprinodontiformes: Aphaniidae)

Eight species are recognised in the Aphanius dispar group. Aphanius dispar from the Red and Mediterranean Sea basins, A. stoliczkanus from coastal areas of the Arabian/Persian Gulf, the northern Arabian Sea east to Gujarat in India, the Gulf of Oman and some endorheic basins in Iran and Pakistan, A. richardsoni from springs in the Dead Sea basin in Jordan and Israel, A. sirhani from the Azraq Oasis in Jordan, A. ginaonis from one spring in Iran, A. furcatus from few streams and springs in Iran and A. stiassnyae from one lake in Ethiopia. Aphanius kruppi, new species, from the Wadi al Batha drainage in northern Oman, is distinguished from adjacent A. stoliczkanus by having 9-14 brown or grey lateral bars on the flank in the male, a roundish, diamond-shaped or somewhat vertically-elongate blotch centred on the caudal-fin base in the female and 2-3 scale rows on the caudal-fin base. The available molecular genetic data for A. dispar reject the hypothesis of the presence of a single widespread coastal species in the Middle East and make it likely that two additional unidentified species occur in the Red Sea basin.