Evidence For Cryptic Species Research Articles

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.

Molecular Species Identification of Astrotoma agassizii from Planktonic Embryos: Further Evidence for a Cryptic Species Complex

The phrynophiurid brittle star Astrotoma agassizii is abundant in the cold temperate Magellanic region of South America and has a circumpolar Antarctic distribution. Three genetically distinct lineages were recently identified, with one in Antarctica geographically and genetically isolated from both South American lineages (Hunter R, Halanych KM. 2008. Evaluating connectivity in the brooding brittle star Astrotoma agassizii across the Drake Passage in the Southern Ocean. J Hered. 99:137-148.). Despite being an apparent brooding species, A. agassizii displayed a high genetic homogeneity at 2 mitochondrial markers (16s and COII) across a geographical range of more than 500 km along the Antarctic Peninsula. Here, using 16s ribosomal RNA sequences, we match a variety of early developmental stages (fertilized eggs, embryos; n = 12) collected from plankton samples in the Ross Sea to sequences of A. agassizii from the Antarctic Peninsula. The single 16s haplotype reported here is an identical match to one 16s haplotype found for A. agassizii from the Antarctic Peninsula, more than 5000 km away. Based on the regular occurrence of A. agassizii developmental stages in plankton samples, we propose that the Antarctic lineage of this species has a planktonic dispersive stage, with brooding restricted to the South American lineages. A different developmental mode would provide further evidence for cryptic speciation in this brittle star.

High divergence across the whole mitochondrial genome in the “pan-Antarctic” springtail Friesea grisea: Evidence for cryptic species?

Collembola are one of the few hexapod groups adapted to live in the harsh environmental conditions of Antarctic terrestrial ecosystems. Diversity is limited to a few species that can be very abundant in coastal deglaciated sites. A remarkable lack of overlap in Collembola species composition is evident between Western and Eastern Antarctica, and Friesea grisea is currently the only species whose distribution is thought to span these two main regions of the continent. However, our analysis of the complete sequences of the mitochondrial genomes from specimens obtained from each of the two regions showed unexpected genetic divergence, well above the average levels observed between populations belonging to the same species, and so indicating that these are actually separate species, despite their lack of distinguishing morphology. Detailed analysis of the two genomes showed the presence of a non-coding region observed between trnS(uga) and nad1. Other features of these mitochondrial genomes, such as base compositional bias, secondary structure features of tRNAs and the presence of regulatory elements in the control region, are described and discussed from an evolutionary standpoint.

Evidence of cryptic speciation in a fossorial reptile: description of a new species of Blanus (Squamata: Amphisbaenia: Blanidae) from the Iberian Peninsula

A recent phylogeographic study using mitochondrial and nuclear DNA revealed the presence of two well defined allopatric clades of Blanus cinereus in the Iberian Peninsula. Using both univariate and multivariate statistical analyses, we show evidence of morphological differentiation between the two clades. Despite the lack of visually diagnosable morphological characters, the morphological and molecular data suggest that differentiation between the two clades was significantly enough to prevent in the past gene flow and therefore to warrant a specific status for each of the two clades. We suggest that the constraints of the subterranean life could increase the chances of experiencing cryptic speciation in worm lizards. In order to establish a taxonomic re-arrangement for the Iberian Blanus we designate a lectotype for B. cinereus from populations of central Iberia and describe a new species; Blanus mariae sp. nov. from southwestern regions of the Iberian Peninsula.

Unusual intra-individual karyotypical variation and evidence of cryptic species in Amazonian populations ofPristimantis(Anura, Terrarana)

We report a cytogenetic analysis of three Pristimantis fenestratus frog populations of the Amazon region in Brazil. The specimens were sampled in Borba and Manaus, Amazonas State, and in Rio Branco, Acre State. They were analyzed using Giemsa, silver staining, C-banding and FISH with rDNA probes. The karyotypes of the three populations revealed 2n = 34 chromosomes, but they differed in the number and position of Ag-NORs and in the heterochromatin pattern as well. The NOR was located on the pairs 05 and 07 in the Rio Branco specimens, pair 10 in the Manaus specimens, and pair 1 in the Borba specimens. A small C-band was detected on the telomeric region of the pair 05 in the Borba population, while in the Manaus there was a heterochromatic block adjacent to the centromere of pair 9. An unusual intra-individual variation of chromosome number was observed in metaphases of Rio Branco specimens, comprising fundamental numbers of 33, 34 and 35. Additionally, interchromosome thread connections were detected between telomere - telomere, centromere - telomere and centromere - centromere regions, and among chromosomal heterochromatin-rich sites. The NOR sites were also involved in those connections. We hypothesize that this variation is due to chromosome missegregation during mitosis. The inter- and intraindividual variation in chromosome number suggests chromosomal instability in P. fenestratus, which has not been detected in any other anuran group. Since Borba is the type-locality of P. fenestratus, a taxonomic review of the Manaus and Rio Branco populations should be done, as indicated by the cytogenetic evidence that they could be new species of Pristimantis.

Ectomycorrhizal Inocybe species associate with the mycoheterotrophic orchid Epipogium aphyllum but not its asexual propagules.

Epipogium aphyllum is a Eurasian achlorophyllous, mycoheterotrophic forest orchid. Due to its rarity, it is often protected, and its biology is poorly known. The identity and pattern of colonization of fungal associates providing carbon to this orchid have not been studied previously. Using samples from 34 individuals from 18 populations in Japan, Russia and France, the following were investigated: (a) colonization patterns of fungal associates of E. aphyllum by microscopy; (b) their identity by PCR amplification of nuclear ribosomal ITS carried out on rhizome fragments and hyphal pelotons. Microscopic investigations revealed that thick rhizomes were densely colonized by fungi bearing clamp-connections and dolipores, i.e. basidiomycetes. Molecular analysis identified Inocybe species as exclusive symbionts of 75 % of the plants investigated and, more rarely, other basidiomycetes (Hebeloma, Xerocomus, Lactarius, Thelephora species). Additionally, ascomycetes, probably endophytes or parasites, were sometimes present. Although E. aphyllum associates with diverse species from Inocybe subgenera Mallocybe and Inocybe sensu stricto, no evidence for cryptic speciation in E. aphyllum was found. Since basidiomycetes colonizing the orchid are ectomycorrhizal, surrounding trees are probably the ultimate carbon source. Accordingly, in one population, ectomycorrhizae sampled around an individual orchid revealed the same fungus on 11.2 % of tree roots investigated. Conversely, long, thin stolons bearing bulbils indicated active asexual multiplication, but these propagules were not colonized by fungi. These findings are discussed in the framework of ecology and evolution of mycoheterotrophy.

Homopholis and blaesodactylus (Squamata: Gekkonidae) revisited: New insights from a molecular phylogeny

South African Homopholis Boulenger, 1885 and Madagascan Blaesodactylus Boettger, 1893 are gekkonid genera originally distinguished by minor differences in scalation. Subsequent morphological studies have argued either for synonymy of the genera, or retention of the original 19th century taxonomy. Moreover, the validity and boundaries of putative species within each genus, as well as the relationships of each genus to other gekkonid genera, remains unresolved and contentious. We sequenced 3433 aligned bp of mitochondrial (ND2 and ND4) and nuclear (RAG‐1 and PDC) DNA sequence data from 14 samples of Homopholis and Blaesodactylus representing all six recognized species in these two genera, and based on a larger, unpublished study of all gekkotan relationships, eight samples of Geckolepis and two species of Afroedura for outgroups. Results indicated a strongly supported, mono‐phyletic clade including Geckolepis as the sister group to Blaesodactylus + Homopholis, with the latter two genera reciprocally monophyletic. Given the clear morphological and biogeographic distinctness of Homopholis sensu stricto from Blaesodactylus, as well as deep genetic divergence between the two groups, we favor the retention of Blaesodactylus as the valid generic name for the Madagascan species. Homopholis mulleri is confirmed as a valid species, and evidence of cryptic speciation is suggested among divergent samples of H. walbergii (often misspelled in the literature), and among samples of Blaesodactylus and Geckolepis. The pattern observed among South African samples of H. walbergii suggests an important role for the Soutpansberg as a barrier to gene flow.

A molecular phylogenetic approach to the phylum Cycliophora provides further evidence for cryptic speciation in Symbion americanus

Cryptic speciation is a phenomenon that has been reported in a wide range of marine invertebrates, including the American cycliophoran Symbion americanus. Although cryptic speciation is often proposed based on phylogeographical analyses, here we demonstrate the utility of multilocus molecular phylogenetic approaches in identifying cryptic lineages within the phylum Cycliophora. Thirteen individuals from three putative cryptic lineages of S. americanus, as well as two individuals each from the European species S. pandora and a new species of Symbion living on Homarus gammarus, were sequenced for up to 4.8 kb of genomic DNA over four loci (COI, 16S rRNA, 18S rRNA and 28S rRNA). Phylogenetic analyses of individual loci as well as combined data clearly support a division between American and European cycliophorans. Moreover, the American cycliophorans consistently form three well‐supported clades, which is congruent with the presence of three putative reproductively isolated lineages in a species complex. Further studies are necessary to more precisely describe the evolution of reproductive isolation within S. americanus.

Is the Scotia Sea a centre of Antarctic marine diversification? Some evidence of cryptic speciation in the circum-Antarctic bivalve Lissarca notorcadensis (Arcoidea: Philobryidae)

The bivalve Lissarca notorcadensis is one of the most abundant species in Antarctic waters and has colonised the entire Antarctic shelf and Scotia Sea Islands. Its brooding reproduction, low dispersal capabilities and epizoic lifestyle predict limited gene flow between geographically isolated populations. Relationships between specimens from seven regions in the Southern Ocean and outgroups were assessed with nuclear 28S rDNA and mitochondrial cytochrome oxidase subunit I (COI) genes. The 28S dataset indicate that while Lissarca appears to be a monophyletic genus, there is polyphyly between the Limopsidae and Philobryidae. Thirteen CO1 haplotypes were found, mostly unique to the sample regions, and two distinct lineages were distinguished. Specimens from the Weddell and Ross Sea form one lineage while individuals from the banks and islands of the Scotia Sea form the other. Within each lineage, further vicariance was observed forming six regionally isolated groups. Our results provide initial evidence for reproductively isolated populations of L. notorcadensis. The islands of the Scotia Sea appear to act as centres of speciation in the Southern Ocean.

Mitochondrial DNA reveals phylogenetic structuring and cryptic diversity in Australian freshwater macroinvertebrate assemblages

Freshwater catchments of south-east Australia possess generally rich and diverse macroinvertebrate faunas, although the genetic structuring of these assemblages is poorly known. In this study, we assessed mitochondrial phylogenetic structure within four genera of macroinvertebrates from the Sydney Water Supply Catchment, south-east Australia: Euastacus (parastacid crayfish), Cheumatopsyche (hydropsychid caddisflies), Atalophlebia (leptophlebiid mayflies) and Paratya (atyid shrimp), with a view to prioritising areas of high diversity for future conservation efforts. We found extremely divergent (≈4–19%) cytochrome c oxidase subunit I (COI) lineages within all surveyed groups, many of which corresponded to recognised taxa, although there was also evidence of cryptic species within three genera; Euastacus, Atalophlebia and Paratya. Distributions of these three genera were associated with high altitude streams, above dam impoundments. Our results have important implications for management of the Sydney Water Supply Catchment. Future disturbance in this region is likely to be high and priority should be directed towards preserving the diversity of fauna in these upland areas. This comparative phylogenetic approach may have value as a means to focus and direct conservation efforts in other areas supporting high biodiversity.

Biochemical Variation in Alpheus (Decapoda, Caridea, Alpheidae) from the Coast of Texas: Evidence for Cryptic Species

Biochemical Variation in Alpheus (Decapoda, Caridea, Alpheidae) from the Coast of Texas: Evidence for Cryptic Species

Wild Lentils

Abstract Wild lentils are potential genetic resources for the cultivated lentil, Lens culinaris ssp. culinaris. Their actual value for crop improvement depends on their genetic relationships with the cultigen and their diversity for traits of economic importance. The current view on Lens taxonomy and the latest information on geographic distribution and ecology of the wild taxa are reviewed. The latter is essential for successful collection of wild lentils in their natural habitats. Intraspecific variation is extensively reviewed and evidence for cryptic speciation has been indicated. Crossability potential divides the genus into two groups: L. culinaris — L. odemensis and L. ervoides — L. nigricans. Crosses between members of different group fail because of hybrid embryo abortion. Using embryo culture, viable hybrids can be obtained between L. ervoides and members of the other group. Of the wild lentils, the putative ancestor of the cultigen, L. culinaris ssp. orientalis, is a member of the crop's primary gene pool, whereas L. odemensis and L. ervoides constitute the secondary gene pool. Morphological, physiological, and genetic attributes of ssp. orientalis have been used to assess the process of lentil domestication. It has been pointed out that elimination of seed dormancy was a necessary step for successful lentil cultivation, and that the dormancy-free type probably evolved in wild stands by the aid of selection pressure exerted by man.

Genetic evidence for cryptic speciation in the marine bryozoan Alcyonidium gelatinosum

The common subtidal marine bryozoan Alcyonidium gelatinosum (L.) is one of the most confusing species of a taxonomically very difficult genus. It shows massive variation for almost all morphological characters. A number of sympatric colonies collected off the coast of Lundy (Bristol Channel) could be clearly divided on morphological grounds into two quite distinct types. Using starch gel electrophoresis the products of a range of genetic enzyme loci were compared between the two morphotypes and also between these and other Bristol Channel populations of A. gelatinosum. Minimal differences were observed between one Lundy morphotype and the other allopatric populations, which it was consequently concluded were conspecific. The second morphotype showed considerable genetic differentiation [genetic identity (Nei, 1972) =0.417] from the other Lundy population, with no common alleles at some loci. The probability of obtaining the observed results by chance in a single population is significantly low (P<10−14). It is therefore concluded that the two morphotypes of A. gelatinosum from Lundy are not conspecific.