Samples of the 'Himantura uarnak' species complex (H. leoparda, H. uarnak, H. undulata under their current definitions), mostly from the Coral Triangle, were analyzed using nuclear markers and mitochondrial DNA sequences. Genotypes at five intron loci showed four reproductively isolated clusters of individuals. The COI sequences showed four major mitochondrial lineages, each diagnostic of a cluster as defined by nuclear markers. No mitochondrial introgression was detected. The average Kimura-2 parameter nucleotide distance separating clades was 0.061-0.120 (net: 0.055-0.114), while the distance separating individuals within a clade was 0.002-0.008. Additional, partial cytochrome-b gene sequences were used to link these samples with previously published sequences of reference specimens of the three nominal species. One of the clusters was identified as H. undulata and another one, as H. uarnak, while two cryptic species were uncovered within the recently-described H. leoparda, challenging the current morphology-based taxonomy of species within the H. uarnak species complex.

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.

The maskray from New Caledonia, Neotrygon trigonoides Castelnau, 1873, has been recently synonymized with the blue-spotted maskray, N. kuhlii (Müller and Henle, 1841), a species with wide Indo-West Pacific distribution, but the reasons for this are unclear. Blue-spotted maskray specimens were collected from the Indian Ocean (Tanzania, Sumatra) and the Coral Triangle (Indonesia, Taiwan, and West Papua), and N. trigonoides specimens were collected from New Caledonia (Coral-Sea). Their partial COI gene sequences were generated to expand the available DNA-barcode database on this species, which currently comprises homologous sequences from Ningaloo Reef, the Coral Triangle and the Great Barrier Reef (Coral-Sea). Spotting patterns were also compared across regions. Haplotypes from the Coral-Sea formed a haplogroup phylogenetically distinct from all other haplotypes sampled in the Indo-West Pacific. No clear-cut geographic composition relative to DNA-barcodes or spotting patterns was apparent in N. kuhlii samples across the Indian Ocean and the Coral Triangle. The New Caledonian maskray had spotting patterns markedly different from all the other samples. This, added to a substantial level of net nucleotide divergence (2.6%) with typical N. kuhlii justifies considering the New Caledonian maskray as a separate species, for which we propose to resurrect the name Neotrygon trigonoides.

Aim To test whether or not fungal communities associated with the widespread seagrass, Syringodium isoetifolium can be differentiated on either side of Wallace's line, a boundary line separating Asian and Australasian fauna. Additionally, we examine whether host multilocus genotype predicts fungal community composition. Location A total of 77 samples were collected from 14 sampling sites spanning the Indonesian archipelago. Methods We sequenced the fungal ITS1 gene using Illumia MiSeq technology and used a clustering‐free Divisive Amplicon Denoising Algorithm to infer ribosomal sequence variants. Data were analysed via non‐metric multidimensional scaling, Mantel tests and permutational multivariate analysis of variance. Binary and quantitative null models were used to determine whether results significantly deviated from random. Host genotype was determined by genotyping at 18 microsatellite loci and standard genetic analysis was performed in the R package APE. Results Significant differences in fungal community composition were detected on either side of Wallace's line (p = <.001 R2 = .040). A significant distance decay of similarity pattern was observed between ribosomal sequence variants and geographical distance (p = .001 R2 = .227) and several fungal ribosomal sequence variants were significantly associated with sampling sites found either east or west of Wallace's line. Main conclusions Fungi are generally considered to have excellent dispersal potentials and marine fungi have the potential to disperse far and wide in an environment that has no obvious barriers to dispersal. Despite this assumed excellent dispersal potential, we show that fungal communities on either side of Wallace's line are significantly different from one another. We speculate that limited dispersal and differences in habitat type are responsible for the observed pattern. Work examining biogeographical patterns in marine fungi is still in its infancy and further research is required to fully understand marine fungal biogeography.

It has been previously established that the Leopard Whipray, Himantura leoparda, consists of two genetically isolated, cryptic species, provisionally designated as 'Cluster 1' and 'Cluster 4' (Arlyza et al., Mol. Phylogenet. Evol. 65 (2013) [1]). Here, we show that the two cryptic species differ by the spotting patterns on the dorsal surface of adults: Cluster-4 individuals tend to have larger-ocellated spots, which also more often have a continuous contour than Cluster-1 individuals. We show that H. leoparda's holotype has the typical larger-ocellated spot pattern, designating Cluster 4 as the actual H. leoparda. The other species (Cluster 1) is described as Himantura tutul sp. nov. on the basis of the nucleotide sequence of a 655-base pair fragment of its cytochrome-oxidase I gene (GenBank accession No. JX263335). Nucleotide synapomorphies at this locus clearly distinguish H. tutul sp. nov. from all three other valid species in the H. uarnak species complex, namely H. leoparda, H. uarnak, and H. undulata. H. tutul sp. nov. has a wide distribution in the Indo-West Pacific, from the shores of eastern Africa to the Indo-Malay archipelago. H. leoparda under its new definition has a similarly wide Indo-West Pacific distribution.

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.

a b s t r a c tExon-primed, intron crossing DNA markers (EPICs) were screened for Mendelian-like allele size polymorphisms in three stingray species (Himantura gerrardi, Neotrygon kuhlii and Taeniura lymna) from the central Indo-West Pacific, where they are commercially exploited. Four to 7 size-polymorphic intron loci were selected in a species, and were subsequently tested as genetic markers of stock structure. Sharp genetic differentiation was observed between populations within each species across the Indo-Malay-Papua archipelago (Weir and Cockerham'sθ-values reaching 0.153-0.557 over a few thousand kilometers). A trend of increasing genetic differentiation with increasing geographic distance was apparent in N. kuhlii, in which populations distant by 3000 km were differentiated by an estimatedθ~0.375. This value was an order of magnitude higher than usually reported in coastal benthic teleost fishes and indicates strong sub-population structure. This is likely, at least partly, a consequence of the sedentary benthic habits of N. kuhlii at all life stages. Because replenishment of overexploited populations of N. kuhlii and two other stingray species from the central Indo-West Pacific is unlikely at ecological timescales, management should be planned at the local geographic scale.

Diagnostic description and geographic distribution of four new cryptic species of the blue-spotted maskray species complex (Myliobatoidei: Dasyatidae; Neotrygon spp.