Stepping up to genome scan allows stock differentiation in the worldwide distributed blue shark Prionace glauca.

The blue shark Prionace glauca is a top predator with one of the widest geographical distributions of any shark species. It is classified as Critically Endangered in the Mediterranean Sea, and Near Threatened globally. Previous genetic studies did not reject the null hypothesis of a single global population. The blue shark was proposed as a possible archetype of the "grey zone of population differentiation," coined to designate cases where population structure may be too recent or too faint to be detected using a limited set of markers. Here, blue shark samples collected throughout its global range were sequenced using a specific RAD method (DArTseq), which recovered 37,655 genome-wide single nucleotide polymorphisms (SNPs). Two main groups emerged, with Mediterranean Sea and northern Atlantic samples (Northern population) differentiated significantly from the Indo-west Pacific samples (Southern population). Significant pairwise FST values indicated further genetic differentiation within the Atlantic Ocean, and between the Atlantic Ocean and the Mediterranean Sea. Reconstruction of recent demographic history suggested divergence between Northern and Southern populations occurred about 500 generations ago and revealed a drastic reduction in effective population size from a large ancestral population. Our results illustrate the power of genome scans to detect population structure and reconstruct demographic history in highly migratory marine species. Given that the management plans of the blue shark (targeted or bycatch) fisheries currently assume panmictic regional stocks, we strongly recommend that the results presented here be considered in future stock assessments and conservation strategies.

Diversity, phylogeny, and historical biogeography of large-eye seabreams (Teleostei: Lethrinidae).

The large-eye seabreams or Monotaxinae is one of two subfamilies in the Lethrinidae, a family of perch-like coral reef fishes. Despite its widespread occurrence and its commercial interest in the tropical Indo-West Pacific (IWP), this subfamily has traditionally been considered a taxonomically difficult group. Based on 268 samples collected from all 15 known large-eye seabream species throughout their distribution ranges, we investigated the taxonomic diversity and phylogenetic relationships in the subfamily. From the results of multiple analyses on four gene markers, we confirmed the monophyly of all four genera in the subfamily (Gnathodentex, Gymnocranius, Monotaxis and Wattsia). We confirmed the occurrence of two species in the genus Monotaxis. We reported 15 delimited species within the most speciose genus Gymnocranius, four of which are potentially new species. The time-calibrated phylogenetic reconstruction enabled us to clarify the evolutionary history of the large-eye seabreams and to infer past patterns of species distribution. The most recent common ancestor to the Monotaxinae likely occurred in the central IWP ca. 32 million years ago. A burst of species diversification likely took place during the Mid- to Late Miocene, coinciding with tectonic change in the central IWP region. This gave rise to most extant lineages in Gymnocranius. The observed geographic distribution patterns in the subfamily most likely point to the central IWP as the area of origin and diversification. This was followed by multiple events of centrifugal range expansion towards either the Indian Ocean or the western Pacific Ocean, or both. Our results thus provide new support for S. Ekman's center-of-origin hypothesis.

Characterization of genome-wide microsatellite markers in rabbitfishes, an important resource for artisanal fisheries in the Indo-West Pacific.

Rabbitfishes are reef-associated fishes that support local fisheries throughout the Indo-West Pacific region. Sound management of the resource requires the development of molecular tools for appropriate stock delimitation of the different species in the family. Microsatellite markers were developed for the cordonnier, Siganus sutor, and their potential for cross-amplification was investigated in 12 congeneric species. A library of 792 repeat-containing sequences was built. Nineteen sets of newly developed primers, and 14 universal finfish microsatellites were tested in S. sutor. Amplification success of the 19 Siganus-specific markers ranged from 32 to 79% in the 12 other Siganus species, slightly decreasing when the genetic distance of the target species to S. sutor increased. Seventeen of these markers were polymorphic in S. sutor and were further assayed in S. luridus, S. rivulatus, and S. spinus, of which respectively 9, 10 and 8 were polymorphic. Statistical power analysis and an analysis of molecular variance showed that subtle genetic differentiation can be detected using these markers, highlighting their utility for the study of genetic diversity and population genetic structure in rabbitfishes.

Phylogeography of the sergeants Abudefduf sexfasciatus and A. vaigiensis reveals complex introgression patterns between two widespread and sympatric Indo-West Pacific reef fishes.

On evolutionary timescales, sea level oscillations lead to recurrent spatio-temporal variation in species distribution and population connectivity. In this situation, applying classical concepts of biogeography is challenging yet necessary to understand the mechanisms underlying biodiversity in highly diverse marine ecosystems such as coral reefs. We aimed at studying the outcomes of such complex biogeographic dynamics on reproductive isolation by sampling populations across a wide spatial range of a species-rich fish genus: the sergeants (Pomacentridae: Abudefduf). We generated a mutlilocus data set that included ten morpho-species from 32 Indo-West Pacific localities. We observed a pattern of mito-nuclear discordance in two common and widely distributed species: Abudefduf sexfasciatus and Abudefduf vaigiensis. The results showed three regional sublineages (Indian Ocean, Coral Triangle region, western Pacific) in A. sexfasciatus (0.6-1.5% divergence at cytb). The other species, A. vaigiensis, is polyphyletic and consists of three distinct genetic lineages (A, B and C) (9% divergence at cytb) whose geographic ranges overlap. Although A. vaigiensis A and A. sexfasciatus were found to be distinct based on nuclear information, A. vaigiensis A was found to be nested within A. sexfasciatus in the mitochondrial gene tree. A. sexfasciatus from the Coral Triangle region and A. vaigiensis A were not differentiated from each other at the mitochondrial locus. We then used coalescent-based simulation to characterize a spatially widespread but weak gene flow between the two species. We showed that these fishes are good candidates to investigate the evolutionary complexity of the discrepancies between phenotypic and genetic similarity in closely related species.

Mitochondrial haplotypes indicate parapatric-like phylogeographic structure in blue-spotted maskray (Neotrygon kuhlii) from the Coral Triangle region.

Phylogeographic structure was investigated in the blue-spotted maskray, Neotrygon kuhlii, focusing on the Coral Triangle region. We used as genetic marker a 519-bp fragment of the cytochrome c-oxidase subunit I (COI) gene, sequenced in a total of 147 individuals from 26 sampling locations. The parsimony network of COI haplotypes was split into seven distinct clades within the Coral Triangle region. Different clades had exclusive but contiguous geographic distributions, indicating parapatric-like phylogeographic structure. Strong genetic differences were also inferred between local populations within a clade, where reciprocal monophyly between geographically adjacent samples was observed on several instances. Nearly 25% of the total molecular variance could be ascribed to differences between geographic samples within a clade, whereas interclade variation accounted for >65% of the total variance. The strong phylogeographic structure observed within a clade can be explained by either sedentarity or female philopatry. We interpret the parapatric distribution of clades as the joint result of 1) expansion from refuge populations at times of low sea level, and 2) possible enhanced competition between individuals from different clades, or assortative mating, or hybrid zones, along lines of secondary contact. The parapatric-like structure uncovered in the present study parallels regional differences at nuclear marker loci, thus pointing to incipient speciation within Coral Triangle N. kuhlii.

The coral sea: physical environment, ecosystem status and biodiversity assets.

The Coral Sea, located at the southwestern rim of the Pacific Ocean, is the only tropical marginal sea where human impacts remain relatively minor. Patterns and processes identified within the region have global relevance as a baseline for understanding impacts in more disturbed tropical locations. Despite 70 years of documented research, the Coral Sea has been relatively neglected, with a slower rate of increase in publications over the past 20 years than total marine research globally. We review current knowledge of the Coral Sea to provide an overview of regional geology, oceanography, ecology and fisheries. Interactions between physical features and biological assemblages influence ecological processes and the direction and strength of connectivity among Coral Sea ecosystems. To inform management effectively, we will need to fill some major knowledge gaps, including geographic gaps in sampling and a lack of integration of research themes, which hinder the understanding of most ecosystem processes.

Phylogeography of Long-spined Sea Urchin Diadema setosum Across the Indo-Malay Archipelago.

Widely distributed, broadcast-spawning Diadema sea urchins have been used as model invertebrate species for studying the zoogeography of the tropical Indo-Pacific. So far, the Indo-Malay archipelago, a wide and geographically complex maritime region extending from the eastern Indian Ocean to the western Pacific Ocean, has been under-sampled. This study aims to fill this sampling gap and uncover the phylogeographic structure of the long-spined sea-urchin D. setosum in the central Indo-West pacific region. D. setosum samples (total N = 718) were collected in 13 sites throughout the Indo-Malay archipelago. We sequenced over 1157 bp of COI gene. The Phylogeographic structure was derived from pairwise ФST estimates using multidimensional scaling and hierarchical clustering analysis; biogeographic hypotheses were tested by AMOVA; genetic relationships between haplotypes were summarised in the form of a minimum-spanning network; and pairwise mismatch distributions were compared to the expectations from demographic and spatial expansion models. All samples from the Indo-West Pacific were of the previously uncovered D. setosum-a lineage. Phylogeographic structure was evident: the Andaman Sea population and the northern New Guinea population were genetically distinct. Subtler but significant haplotype-frequency differences distinguished two populations within the Indonesian seas, distributed in a parapatric-like fashion. The phylogeographic partition observed was insufficiently explained by previous biogeographic hypotheses. The haplotype network showed a series of closely related star-shaped haplogroups with a high proportion of singletons. Nucleotide-pairwise mismatch patterns in the two populations from the Indonesian seas were consistent with both demographic and spatial expansion models. While geographic barriers to gene flow were inferred at the western and eastern extremities of the Indo-Malay archipelago, the subtler parapatric pattern observed within the Indonesian seas indicated restriction in gene flow, in a fashion that can hardly be explained by geographic isolation given the dynamic current systems that cross this region. Our results thus raise the hypothesis of subtle reproductive isolation between ecologically incompatible populations. While the coalescence pattern of the Andaman-Sea population suggested demographic stability over evolutionary timescales, that of the two populations from the Indonesian seas indicated recent population expansion, possibly linked to the rapid changes in available D. setosum habitat caused by sea-level oscillations in the late Pleistocene. The phylogeographic patterns observed in this study point to likely allopatric differentiation in the central Indo-West Pacific region. Genetic differences between populations were likely reinforced during interglacials by some form of reproductive isolation.

[Shark attacks in New Caledonia from 1958 to 2020: a review of cases]. / Attaques de requins en Nouvelle-Calédonie de 1958 à 2020 : revue de cas.

Background and objectives: Recent shark attacks in New Caledonia have prompted local authorities to elaborate a risk-management plan. The objective of the present paper is to produce detailed data on shark attacks that occurred in New Caledonian waters for the last few decades, as well as on the injuries of the victims, in order to inform rescue and medical services as well as authorities in charge of educating the public and providing security. Methods: Incidents involving sharks and humans in New Caledonia for the last six decades were included into a database. Sharks were tentatively identified to species according to the shape, size and other external characteristics of injuries to the victims, together with witness accounts. The severity of shark bites was evaluated against the scale proposed by A.K. Lentz and co-authors (Am Surg. 2010;76:101-6). Results: Sixty-seven shark-attack cases were recorded in New Caledonia from 1958 to 2020, of which 13 were lethal. The majority of the attacks concerned spearfishers and freedivers collecting invertebrates (58.5% of total). In the last decades, shark attacks may have increased towards bathers, swimmers and snorkelers (18.5%), and people taking part in water sports including surf, kitesurf, windsurf and SUP foil (14%). One scuba diver was also attacked (1.5%). Twenty attacks including 8 lethal ones were ascribed to the tiger shark Galeocerdo cuvier; 14 attacks including 2 lethal ones to the bull shark Carcharhinus leucas; 2 attacks including 1 lethal to the great white shark Carcharodon carcharías. The lethality of attacks was almost one in five, above the global average. Conclusions: Feeding incentive appeared to be a frequent factor triggering attacks. The education of the public should be promoted as a preventive measure aiming to reduce the risk of such accidents.

Characterization of mitochondrial genome of Indian Ocean blue-spotted maskray, Neotrygon indica and its phylogenetic relationship within Dasyatidae Family.

The present study characterized complete mitochondrial genome of Blue-spotted maskray, Neotrygon indica and studied the evolutionary relationship of the species within the Dasyatidae family. The total length of the mitogenome was 17,974 bp including 37 genes and a non-coding control region. The average frequency of nucleotides in protein-coding genes was A: 29.1 %, T: 30.2 %, G: 13.0 % and C: 27.7 % with AT content of 59.3 %. The values of AT and GC skewness were -0.018 and -0.338, respectively. Comparative analyses showed a large number of average synonymous substitutions per synonymous site (Ks) in gene NADH4 (5.07) followed by NADH5 (4.72). High values of average number of non-synonymous substitutions per non-synonymous site (Ka) were observed in genes ATPase8 (0.54) and NADH2 (0.44). Genes NADH4L and NADH2 showed high interspecific genetic distance values of 0.224 ± 0.001 and 0.213 ± 0.002, respectively. Heat map analysis showed variation in codon usage among different species of the Dasyatidae family. The phylogenetic tree showed a sister relationship between the Dasyatinae and the Neotrygoninae subfamilies. Neotrygon indica formed as a sister species to the clade consisting of N. varidens and N. orientalis. Based on the present results, Neotrygon indica could have diverged from the common ancestor of the two latter in the Plio-Pleistocene. The present study showed distinct characteristics of N. indica from its congeners through comparative mitogenomics.

Biodiversity inventory of the grey mullets (Actinopterygii: Mugilidae) of the Indo-Australian Archipelago through the iterative use of DNA-based species delimitation and specimen assignment methods.

DNA barcoding opens new perspectives on the way we document biodiversity. Initially proposed to circumvent the limits of morphological characters to assign unknown individuals to known species, DNA barcoding has been used in a wide array of studies where collecting species identity constitutes a crucial step. The assignment of unknowns to knowns assumes that species are already well identified and delineated, making the assignment performed reliable. Here, we used DNA-based species delimitation and specimen assignment methods iteratively to tackle the inventory of the Indo-Australian Archipelago grey mullets, a notorious case of taxonomic complexity that requires DNA-based identification methods considering that traditional morphological identifications are usually not repeatable and sequence mislabeling is common in international sequence repositories. We first revisited a DNA barcode reference library available at the global scale for Mugilidae through different DNA-based species delimitation methods to produce a robust consensus scheme of species delineation. We then used this curated library to assign unknown specimens collected throughout the Indo-Australian Archipelago to known species. A second iteration of OTU delimitation and specimen assignment was then performed. We show the benefits of using species delimitation and specimen assignment methods iteratively to improve the accuracy of specimen identification and propose a workflow to do so.

Pleistocene separation of mitochondrial lineages of Mytilus spp. mussels from Northern and Southern Hemispheres and strong genetic differentiation among southern populations.

Smooth-shelled mussels, Mytilus spp., have an antitropical distribution. In the Northern Hemisphere, the M. edulis complex of species is composed of three genetically well delineated taxa: M. edulis, M. galloprovincialis and M. trossulus. In the Southern Hemisphere, morphological characters, allozymes and intron length polymorphisms suggest that Mytilus spp. populations from South America and Kerguelen Islands are related to M. edulis and those from Australasia to M. galloprovincialis. On the other hand, a phylogeny of the 16S rDNA mitochondrial locus demonstrates a clear distinctiveness of southern mussels and suggests that they are related to Mediterranean M. galloprovincialis. Here, we analysed the faster-evolving cytochrome oxidase subunit I locus. The divergence between haplotypes of populations from the two hemispheres was confirmed and was found to predate the divergence between haplotypes of northern M. edulis and M. galloprovincialis. In addition, strong genetic structure was detected among the southern samples, revealing three genetic entities that correspond to (1) South America and Kerguelen Island, (2) Tasmania, (3) New Zealand. Using the trans-Arctic interchange as a molecular clock calibration, we estimated the time since divergence of populations from the two hemispheres to be between 0.5 million years (MY) and 1.3 MY (average 0.84 MY). The contrasting patterns observed for the nuclear and the organelle genomes suggested two alternative, complex scenarios: two trans-equatorial migrations and the existence of differential barriers to mitochondrial and nuclear gene flow, or a single trans-equatorial migration and a view of the composition of the nuclear genome biased by taxonomic preconception.

Cryptic genetic diversity in the mottled rabbitfish Siganus fuscescens with mitochondrial introgression at a contact zone in the South China Sea.

The taxonomy of the mottled rabbitfish Siganus fuscescens species complex has long been challenging. In this study, we analyzed microsatellite genotypes, mitochondrial lineages, and morphometric data from 373 S. fuscescens individuals sampled from the northern Philippines and Hong Kong (South China Sea, Philippine Sea and Sulu Sea basins), to examine putative species boundaries in samples comprising three co-occurring mitochondrial lineages previously reported to characterize S. fuscescens (Clade A and Clade B) or S. canaliculatus (Clade C). We report the existence of two cryptic species within S. fuscescens in the northeast region of the South China Sea and northern Philippine Sea, supported by genetic and morphological differences. Individual-based assignment methods recovered concordant groupings of individuals into two nuclear genotype clusters (Cluster 1, Cluster 2) with (1) limited gene flow, if any, between them (FST = 0.241; P < 0.001); (2) low frequency of later-generation hybrids; (3) significant association with mitochondrial Clade A and Clade B, respectively; and (4) subtle yet significant body shape differences as inferred from geometric morphometric analysis. The divergence between mitochondrial Clade C and the two other clades was not matched by genetic differences at microsatellite marker loci. The occurrence of discordant mitonuclear combinations (20.5% of the total number of individuals) is thought to result from mitochondrial introgression, consistent with a scenario of demographic, and presumably spatial, post-Pleistocene expansion of populations from northern regions into a secondary contact zone in the South China Sea. Mitonuclear discordance due to introgression obscures phylogenetic relationships for recently-diverged lineages, and cautions against the use of mitochondrial markers alone for species identification within the mottled rabbitfish species complex in the South China Sea region.

Neotrygon indica sp. nov., the Indian Ocean blue-spotted maskray (Myliobatoidei, Dasyatidae).

The blue-spotted maskray, previously N. kuhlii, consists of up to eleven lineages representing separate species. Nine of these species (N. australiae, N. bobwardi, N. caeruleopunctata, N. malaccensis, N. moluccensis, N. orientale, N. vali, N. varidens, N. westpapuensis) have already been formally described and two (Indian Ocean maskray and Ryukyu maskray) remain undescribed. Here, the Indian Ocean maskray is described as a new species, Neotrygon indica sp. nov. Specimens of the new species were generally characterized on their dorsal side by a moderately large number of small ocellated blue spots, a low number of medium-sized ocellated blue spots, the absence of large ocellated blue spots, a high number of dark speckles, a few dark spots, and a conspicuous occipital mark. The new species formed a distinct haplogroup in the tree built from concatenated nucleotide sequences at the CO1 and cytochrome b loci. A diagnosis based on colour patterns and nucleotide sequences at the CO1 and cytochrome b loci is proposed. The distribution of N. indica sp. nov. includes the Indian coast of the Bay of Bengal, the Indian coast of the Laccadives Sea, and Tanzania. Considerable sampling effort remains necessary for an in-depth investigation of the phylogeographic structure of the Indian Ocean maskray.

Cytochrome b barcoding, molecular systematics and geographic differentiation in rabbitfishes (Siganidae).

The fish genus Siganus (Siganidae) is widely distributed in the coastal habitats of all the tropical Indo-Pacific, with 28 nominal species recognized so far, based on general morphology and coloration patterns. A mitochondrial phylogeny of 16 Siganidae species, based on the partial nucleotide sequences of the cytochome b gene, was produced. Individual haplotypes of given nominal species generally clustered at the extremity of long branches, thus validating the current taxonomy. However, S. lineatus haplotypes formed a paraphyletic group including S. guttatus, while S. fuscescens haplotypes were apparently splitted in two groups, calling for further investigation. S. woodlandi and S. argenteus formed a monophyletic group, as expected from their close morphological relatedness, although they were separated by a substantial, 14.5-16.3% nucleotide distance. Among eight species sampled from different locations across the Indo-West Pacific, S. argenteus and S. spinus showed the lowest degree of geographic differentiation, a result that correlated well with their extended pelagic larval stage. Fixation index estimates were high in all six other species tested (S. doliatus, S. fuscescens, S. lineatus, S. puellus, S. punctatus, S. vulpinus). The cytochrome b gene fragment chosen here proved useful as a barcode in Siganidae.

Gymnocranius obesus, a new large-eye seabream from the Coral Triangle.

Two previously recorded new species of the large-eye seabream genus Gymnocranius (Gymnocranius sp. D and Gymnocranius sp. E) remain undescribed. Here we describe Gymnocranius sp. E as Gymnocranius obesus sp. nov. This new species is morphologically distinct from all other known species under Gymnocranius by the following combination of characters: relatively deep body, with ratio of standard length to body depth 2.2-2.4; protruding large eye, with eye diameter about equal to or slightly larger than inter-orbital width; caudal fin moderately forked; no blue spots or wavy blue lines on cheek and snout in adults; fourth transversal dark bar on flank running from the sixth spine of the dorsal fin to the origin of the anal fin; anal, caudal and dorsal fins drab with yellowish to yellow margins. Gymnocranius obesus sp. nov. is distinct from G. griseus, with which it has been previously confused by a relatively larger head, scales above lateral line without dark basal patch, and a smaller number of front scales on the dorsal side of the head. Gymnocranius obesus sp. nov. is genetically distinct from its closest known relative, Gymnocranius sp. D by 104 diagnostic nucleotide characters, which translates into a 9.6% sequence divergence at the mitochondrial cytochrome b gene. Gymnocranius obesus sp. nov. reaches a length of at least 295mm. Its distribution, from the Ryukyu Islands to Bali, including Taiwan and the Flores Sea, mostly coincides with the western half of the Coral Triangle.

Marine mammal strandings in the New Caledonia region, Southwest Pacific.

Four hundred twenty three marine mammals, in 72 stranding events, were recorded between 1877 and 2005 in New Caledonia, the Loyalty Islands, and Vanuatu in the southwest Pacific. Sixteen species were represented in this count, including: minke whale, Balaenoptera acutorostrata (1 single stranding), sei whale, B. borealis (1 single stranding), blue whale, B. musculus (1 single stranding), humpback whale, Megaptera novaeangliae (2 single strandings), giant sperm whale, Physeter macrocephalus (18 single strandings, 2 pair strandings), pygmy sperm whale, Kogia breviceps (5 single strandings), dwarf sperm whale, K. sima (2 single strandings, 1 triple stranding), Blainville's beaked whale, Mesoplodon densirostris (2 single strandings), short-finned pilot whale, Globicephala macrorhynchus (4 strandings, 56 individuals), melon-headed whale, Peponocephala electra (1 single stranding and 2 mass strandings totalling 231 individuals), common dolphin, Delphinus delphis (1 single stranding), spinner dolphin, Stenella longirostris (1 pair stranding and 2 mass strandings of groups of approximately 30 individuals each), Indian Ocean bottlenose dolphin, Tursiops aduncus (2 single strandings), dugong, Dugong dugon (14 single strandings), and New Zealand fur seal, Arctocephalus forsteri (3 single strandings). A stranded rorqual identified as an Antarctic minke whale (B. bonaerensis), with coloration patterns that did not match known descriptions, was also reported. Sei whale was recorded for the first time in the tropical Southwest Pacific region and Antarctic minke whale, melon-headed whale, and Indian Ocean bottlenose dolphin were recorded for the first time in New Caledonia. Strandings of sperm whales were most frequent in the spring, but also occurred in autumn months, suggesting a seasonal pattern of occurrence possibly related to seasonal migration. One stranded humpback whale bore the scars of a killer whale's attack and one dugong was injured by a shark. Scars left by propellers were noted on several stranded animals including one Antarctic minke whale, one pygmy sperm whale, one dwarf sperm whale, and four dugongs. Collisions with vessels were suspected to be a frequent cause of death for dugong.

Multiple cryptic species in the blue-spotted maskray (Myliobatoidei: Dasyatidae: Neotrygon spp.): An update.

Previous investigations have uncovered divergent mitochondrial clades within the blue-spotted maskray, previously Neotrygon kuhlii (Müller and Henle). The hypothesis that the blue-spotted maskray may consist of a complex of multiple cryptic species has been proposed, and four species have been recently described or resurrected. To test the multiple cryptic species hypothesis, we investigated the phylogenetic relationships and coalescence patterns of mitochondrial sequences in a sample of 127 new individuals from the Indian Ocean and the Coral Triangle region, sequenced at both the CO1 and cytochrome b loci. The maximum-likelihood (ML) tree of concatenated CO1+cytochrome b gene sequences, rooted by the New Caledonian maskray N. trigonoides, yielded 9 strongly supported, main clades. Puillandre's ABGD algorithm detected gaps in nucleotide distance consistent with the ML phylogeny. The general mixed Yule-coalescent algorithm partitioned the dataset into putative species generally consistent with the ML phylogeny. Nuclear markers generally confirmed that distinct mitochondrial clades correspond to genetically isolated lineages. The nine main lineages identified by ML analysis were geographically distributed in a parapatric fashion, indicating reproductive isolation. The hypothesis of multiple cryptic species is thus validated.