Himantura tutul sp. nov. (Myliobatoidei: Dasyatidae), a new ocellated whipray from the tropical Indo-West Pacific,described from its cytochrome-oxidase I gene sequence

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.     

Genetic diversity of <Emphasis Type="Italic">Haemaphysalis qinghaiensis</Emphasis> (Acari: Ixodidae) in western China

Haemaphysalis qinghaiensis as an endemic species in China mainly infests domestic animals and causes great harm to animals and humans in Northwestern plateau. However, there is no information about genetic diversity within the recently established populations of this tick species. Therefore, the present study analyzed the fragments of mitochondrial 16S rDNA, COI and the nuclear gene ITS1 of 56 H. qinghaiensis ticks across four regions of China which are main endemic areas of this species. Analysis showed 98.1–100% (16S rDNA), 97.9–100% (COI), 99.7–100% (ITS1) identity within individuals. For these sequences, 9, 15 and 8 haplotypes were found for 16S rDNA, COI and ITS1, respectively. Ticks from Yongjing were the most variable group, followed by Lintan, Huangyuan, and Tianzhu. Based on parallel analysis of the mitochondrial and nuclear genetic diversity of H. qinghaiensis, our results indicated that mitochondrial markers (especially COI) were much more useful than nuclear ITS for intraspecific genetic variability analysis.     

Population Genetic Structure and Potential Incursion Pathways of the Bluetongue Virus Vector Culicoides brevitarsis (Diptera: Ceratopogonidae) in Australia

Culicoides brevitarsis is a vector of the bluetongue virus (BTV), which infects sheep and cattle. It is an invasive species in Australia with an assumed Asian/South East Asian origin. Using one mitochondrial marker (i.e., part of the cytochrome oxidase subunit I gene) and six nuclear markers, we inferred population genetic structure and possible incursion pathways for Australian C. brevitarsis. Nine mitochondrial haplotypes, with low nucleotide sequence diversity (0.0–0.7%) among these, were identified in a sample of 70 individuals from seven sites. Both sets of markers revealed a homogeneous population structure, albeit with evidence of isolation by distance and two genetically distinct clusters distributed along a north-to-south cline. No evidence of a cryptic species complex was found. The geographical distribution of the mitochondrial haplotypes is consistent with at least two incursion pathways into Australia since the arrival of suitable livestock hosts. By contrast, 15 mitochondrial haplotypes, with up to four times greater nucleotide sequence diversity (0.0–2.9%) among these, were identified in a sample of 16 individuals of the endemic C. marksi (sampled from a site in South Australia and another in New South Wales). A phylogenetic tree inferred using the mitochondrial marker revealed that the Australian and Japanese samples of C. brevitarsis are as evolutionarily different from one another as some of the other Australian species (e.g., C. marksi, C. henryi, C. pallidothorax) are. The phylogenetic tree placed four of the species endemic to Australia (C. pallidothorax, C. bundyensis, C. marksi, C. henryi) in a clade, with a fifth such species (C. bunrooensis) sharing a common ancestor with that clade and a clade comprising two Japanese species (C. verbosus, C. kibunensis).     

Implications of Hybridization,NUMTs, and Overlooked Diversity for DNA Barcoding of Eurasian Ground Squirrels

The utility of DNA Barcoding for species identification and discovery has catalyzed a concerted effort to build the global reference library; however, many animal groups of economical or conservational importance remain poorly represented. This study aims to contribute DNA barcode records for all ground squirrel species (Xerinae, Sciuridae, Rodentia) inhabiting Eurasia and to test efficiency of this approach for species discrimination. Cytochrome c oxidase subunit 1 (COI) gene sequences were obtained for 97 individuals representing 16 ground squirrel species of which 12 were correctly identified. Taxonomic allocation of some specimens within four species was complicated by geographically restricted mtDNA introgression. Exclusion of individuals with introgressed mtDNA allowed reaching a 91.6% identification success rate. Significant COI divergence (3.5–4.4%) was observed within the most widespread ground squirrel species (Spermophilus erythrogenys, S. pygmaeus, S. suslicus, Urocitellus undulatus), suggesting the presence of cryptic species. A single putative NUMT (nuclear mitochondrial pseudogene) sequence was recovered during molecular analysis; mitochondrial COI from this sample was amplified following re-extraction of DNA. Our data show high discrimination ability of 100 bp COI fragments for Eurasian ground squirrels (84.3%) with no incorrect assessments, underscoring the potential utility of the existing reference librariy for the development of diagnostic ‘mini-barcodes’.     

A new quadrannulate species of Orobdella (Hirudinida,Arhynchobdellida, Orobdellidae) from central Honshu,Japan

A new quadrannulate species of Orobdella, Orobdella masaakikuroiwai sp. n., from the mountainous region of central Honshu, Japan is described. This is only the second small species known within this genus, with a body length of less than 4 cm for mature individuals. Phylogenetic analyses using nuclear 18S rDNA and histone H3 as well as mitochondrial COI, tRNA^Cys, tRNA^Met, 12S, tRNA^Val, 16S, and ND1 markers showed that Orobdella masaakikuroiwai sp. n. is the sister species of the quadrannulate Orobdella whitmani Oka, 1895. Phylogenetic relationships within Orobdella masaakikuroiwai sp. n. conducted using mitochondrial markers reveled a distinction between eastern and western phylogroups.     

Determination of evolutionary units in European representatives of the crab genus Pilumnus

Bristle crabs of the genus Pilumnus (Brachyura: Heterotremata: Pilumnidae) are common inhabitants of European waters. They are easily identifiable as a genus, but with the exception of P. inermis, intrageneric classification turns out to be quite complex. There is no general agreement on the number and distinction of species. Therefore, this genus is well-suited for comparative molecular studies. Specimens of the Pilumnus hirtellus complex, here defined as including Pilumnus hirtellus, P. villosissimus, P. spinifer, P. aestuarii, and an undescribed species, were gathered from throughout the Mediterranean Sea and the eastern Atlantic Ocean. DNA sequence data were obtained from the barcoding region of the cytochrome oxidase 1 mitochondrial gene and used for reconstruction of a phylogenetic tree and a haplotype network. The morphology of the gastric ossicles was compared in the search of separating characters. Our results give evidence for five genetic clusters within the P. hirtellus complex. There is negligible geographic variation within these clusters. Unambiguous mtDNA sequences within morphologically variable local populations argue against possible hybridization. The here encountered evolutionary units are relatively young and possibly allow to study ongoing processes of morphological, genetic, and ecological differentiation, leading to speciation and radiations in the coastal marine environment.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

Microsatellite markers uncover cryptic species of Odontotermes (Termitoidae: Termitidae) from Peninsular Malaysia

Termites from the genus Odontotermes are known to contain numerous species complexes that are difficult to tell apart morphologically or with mitochondrial DNA sequences. We developed markers for one such cryptic species complex, that is, Odontotermes srinakarinensis sp. nov. from Maxwell Hill Forest Reserve (Perak, Malaysia), and characterised them using a sample of 41 termite workers from three voucher samples from the same area. We then genotyped 150 termite individuals from 23 voucher samples/colonies of this species complex from several sites in Peninsular Malaysia. We analysed their population by constructing dendograms from the proportion of shared-alleles between individuals and genetic distances between colonies; additionally, we examined the Bayesian clustering pattern of their genotype data. All methods of analysis indicated that there were two distinct clusters within our data set. After the morphologies of specimens from each cluster were reexamined, we were able to separate the two species morphologically and found that a single diagnostic character found on the mandibles of its soldiers could be used to separate the two species quite accurately. The additional species in the clade was identified as Odontotermes denticulatus after it was matched to type specimens at the NHM London and Cambridge Museum of Zoology.     

Phylogenetic Relationships among Holarctic Populations of Chironomus entis and Chironomus plumosus in View of Possible Horisontal Transfer of Mitochondrial Genes

In eight Holarctic populations of two typical chironomid sibling species of the plumosus group, Chironomus entisandChironomus plumosus, nucleotide sequences of mitochondrial (cytb) and nuclear (gb2b) gene regions were examined. The phylogenetic trees reflecting the evolutionary histories of the nuclear and mitochondrial markers exhibited significant differences. On the tree based on the nuclear gene sequences the populations clustered according to their species affiliation, whereas on the tree based on the mitochondrial gene sequences the populations were grouped according to their geographic position. This discrepancy is probably explained by mitochondrial gene flow between sympatric species with incomplete reproductive isolation (sibling species). Based on our results together with the earlier data on nuclear and mitochondrial gene sequences of some other species from the phylogenetic group plumosus, a scheme of phylogenetic relationships within this group is proposed. This scheme is in many ways different from the traditional view on the evolutionary relationships among species of the plumosus group.     

Patterns of genetic structure among Hawaiian corals of the genus Pocillopora yield clusters of individuals that are compatible with morphology

Six variable sequence markers are developed and analyzed to find out species boundaries in Hawaiian corals of the genus Pocillopora: the putative mitochondrial control region; a recently discovered, hypervariable mitochondrial open reading frame; the internal transcribed spacer 2 (ITS2), located in the nuclear ribosomal DNA; three nuclear introns of calmodulin, elongation factor-1alpha and the ATP synthase beta subunit. Using the first two markers, we identify five distinct mitochondrial lineages and these lineages are compatible with morphology. The situation is more complex with nuclear markers since more than two haplotypes are observed in some individuals. To detect clusters of individuals, haplotype networks are constructed with additional connections drawn between co-occurring haplotypes to delineate potential fields for recombination: few clusters of nuclear haplotypes are found to correspond to clusters of individuals, but those that are detected (mostly in the ITS2 dataset) are also compatible with morphology.     

Morphological and mitochondrial DNA analyses indicate the presence of a hybrid zone between two species of leaf beetle (Coleoptera; Chrysomelidae) in Southern Spain

The present study reports a case where the survey of morphological and mitochondrial DNA variation among populations of a species complex of leaf beetle, the Gonioctena variabilis complex, has lead to the identification of a hybrid zone between two species of the complex in Southern Spain. The complex is divided into four species distributed around the western Mediterranean region. The four species, G. variabilis, Gonioctena aegrota, Gonioctena gobanzi, and Gonioctena pseudogobanzi, are traditionally determined by differences in the morphology of the male genitalia (aedeagus). To gain insight into the history of the speciation process within this species complex, we sampled populations in Portugal, Spain, Southern France, and Northern Italy. We sequenced a portion of the mitochondrial control region of each individual collected. A haplotype network of these sequences was found to comprise four distinct groups of sequence types, separated by a relatively large number of mutations. Moreover, in most of the samples for which morphological and molecular variation is available, there is a one‐to‐one correspondence between haplotype group, defined by mitochondrial sequence variation, and morphological groups defined on the basis of the aedeagus, showing evidence of four historically independent evolutionary units. This supports the use of the aedeagus morphology as a taxonomically informative trait in this species complex and a recent taxonomic revision upgrading four formerly subspecies, corresponding to the evolutionary units identified in the present study, to species status. However, some of the individuals from our samples in Southern Spain, morphologically identified as G. aegrota, were found to possess mitochondrial sequences typical of G. pseudogobanzi. The opposite case was also found. This suggests the presence of a zone of contact and hybridization between G. aegrota and G. pseudogobanzi. The location of this hybrid zone appears to be unusual. We identify historical scenarios that may explain our observations. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94 , 105–114.     

DNA barcoding provides distinction between Radix Astragali and its adulterants

Based on variable nuclear and/or organellar DNA sequences among vastly divergent species as well as morphologically indistinguishable species, DNA barcoding is widely applicable in species identification, biodiversity studies, forensic analyses, and authentication of medicinal plants. The roots of Astragalus membranaceus and A. membranaceus var. mongholica are commonly used as Radix Astragali in several Asian countries, including China, Japan, and Korea. However, in addition to the two species recorded in the Chinese Pharmacopoeia, there are twenty-three species from different genera including Astragalus, Oxytropis, Hedysarum, and Glycyrrhiza, which have been used as adulterants not only in trading markets but also by the herbal medicine industry. Therefore, a simple, reliable, and accurate classification method is important for distinguishing authentic Radix Astragali from its adulterants. In this study, we acquired data for 37 samples from four related genera within the family Fabaceae. Then we compared four candidate DNA barcoding markers using ITS, matK, rbcL, and coxI sequences from nuclear, chloroplast, and mitochondrial genomes, all commonly used for plants to identify genetic variations among genera, intraspecies, and interspecies. We observed higher divergences among genera and interspecies for ITS, which have the average Kimura 2-parameter distances of 4.5% and 14.1%, respectively, whereas matK was found to have sufficient divergence at the intraspecific level. Moreover, two indels detected in the matK sequence are useful for PCR studies in distinguishing Radix Astragali from its adulterants. This study suggests that the combined barcoding regions of ITS and matK are superior barcodes for Radix Astragali and further studies should focus on evaluating the applicability and accuracy of such combined markers for a wide range of traditional Chinese herbs.     

Growth of Tropical dasyatid Rays Estimated Using a Multi-Analytical Approach

We studied the age and growth of four sympatric stingrays: reticulate whipray, Himanutra uarnak (n=19); blue mask, Neotrygon kuhlii (n=34); cowtail, Pastinachus atrus (n=32) and blue-spotted fantail, Taeniura lymma (n=40) rays at Ningaloo Reef, a fringing coral reef on the north-western coast of western Australia. Age estimates derived from band counts within sectioned vertebrae ranged between 1 and 27 years (H. uarnak, 1 - 25 yrs.; N. kuhlii, 1.5 - 13 yrs.; P. atrus, 1 - 27 yrs. and T. lymma, 1 -11 yrs.). Due to limitations of sample sizes, we combined several analytical methods for estimating growth parameters. First, we used nonlinear least squares (NLS) to identify the growth model that best fitted the data. We then used this model, prior information and the data within a Bayesian framework to approximate the posterior distribution of the growth parameters. For all species the two-parameter von Bertalanffy growth model provided the best fit to size-at-age datasets. Based on this model, the Bayesian approach allowed the estimation of median values of W _D∞ (cm) and k (yr^-1) for the four species (H. uarnak: 149 and 0.12; N. kuhlii: 42 and 0.38; P. atrus 156 and 0.16, and T. lymma 33 and 0.24, respectively). Our approach highlights the value of combining different analytical methods and prior knowledge for estimating growth parameters when data quality and quantity are limited.     

Rearing trials of Halla parthenopeia under laboratory conditions (Polychaeta: Oenonidae)

A small scale attempt to maintain and rear the worm Halla parthenopeia in laboratory conditions was conducted. Five bivalve species (Paphia undulata, Cerastoderma glaucum, Venerupis pullastra, Ruditapes decussata, and Gafrarium pectinatum) were used to investigate preferred food item, feeding rate and growth of the worm. Hallaparthenopeia are capable of using different types of clams, although they grew better with Paphia undulata and C.glaucum as food items. The highest average daily predation rates in case of P. undulata as a prey were 1.73, 2.13 and 2.57 individuals per predator per day for small, medium and large groups of H. parthenopeia, respectively. The daily predation rate on C. glaucum was low with an average: 0.50, 0.63 and 0.73 individuals per predator per day for the small, medium and large worm groups, respectively. The daily growth rate of H. parthenopiea increased when it was fed P. undulata (average: 0.083, 0.071 and 0.038 g/day for small, medium and large worm groups respectively), compared to an average of 0.044, 0.034 and 0.020 g/day for small, medium and large worm groups, respectively, when worms were fed with C. glaucum. The biochemical composition of three different sizes of the worm was also determined. Protein was the highest biochemical constituent with an average of 51% of the dry weight, followed by lipids with an average of 25.88% of the dry weight; meanwhile carbohydrate was present at an average of 20.72%. Our findings indicate that growth of H. parthenopeia can be improved when fed with a suitable prey item and suggest that it is feasible to successfully culture this protein-rich worm in captivity.     

Molecular signatures of divergence and selection in closely related pine taxa

Efforts to detect loci under selection in plants have mostly focussed on single species. However, assuming that intraspecific divergence may lead to speciation, comparisons of genetic variation within and among recently diverged taxa can help to locate such genes. In this study, coalescent and outlier detection methods were used to assess nucleotide polymorphism and divergence at 79 nuclear gene fragments (1212 SNPs) in 16 populations (153 individuals) of the closely related, but phenotypically and ecologically distinct, pine taxa Pinus mugo, P. uliginosa and P. uncinata across their European distributions. Simultaneously, mitochondrial DNA markers, which are maternally inherited in pines and distributed by seeds at short geographic distance, were used to assess genetic relationships of the focal populations and taxa. The majority of nuclear loci showed homogenous patterns of variation between the taxa due to a high number of shared SNPs and haplotypes, similar levels of polymorphism, and low net divergence. However, against this common genetic background and an overall low population structure within taxa at mitochondrial markers, we identified several genes showing signatures of selection, accompanied by significant intra- and interspecific divergence. Our results indicate that loci involved in species divergence may be involved in intraspecific local adaptation.     

Sea surface temperature,rather than land mass or geographic distance,may drive genetic differentiation in a species complex of highly dispersive seabirds

Seabirds, particularly Procellariiformes, are highly mobile organisms with a great capacity for long dispersal, though simultaneously showing high philopatry, two conflicting life‐history traits that may lead to contrasted patterns of genetic population structure. Landmasses were suggested to explain differentiation patterns observed in seabirds, but philopatry, isolation by distance, segregation between breeding and nonbreeding zones, and oceanographic conditions (sea surface temperatures) may also contribute to differentiation patterns. To our knowledge, no study has simultaneously contrasted the multiple factors contributing to the diversification of seabird species, especially in the gray zone of speciation. We conducted a multilocus phylogeographic study on a widespread seabird species complex, the little shearwater complex, showing highly homogeneous morphology, which led to considerable taxonomic debate. We sequenced three mitochondrial and six nuclear markers on all extant populations from the Atlantic (lherminieri) and Indian Oceans (bailloni), that is, five nominal lineages from 13 populations, along with one population from the eastern Pacific Ocean (representing the dichrous lineage). We found sharp differentiation among populations separated by the African continent with both mitochondrial and nuclear markers, while only mitochondrial markers allowed characterizing the five nominal lineages. No differentiation could be detected within these five lineages, questioning the strong level of philopatry showed by these shearwaters. Finally, we propose that Atlantic populations likely originated from the Indian Ocean. Within the Atlantic, a stepping‐stone process accounts for the current distribution. Based on our divergence time estimates, we suggest that the observed pattern of differentiation mostly resulted from historical and current variation in sea surface temperatures.     

Description of a new species of Moniliformis (Acanthocephala: Moniliformidae) from Peromyscus hylocetes (Rodentia: Cricetidae) in Mexico

Moniliformis ibunami n. sp., is described from the intestine of the transvolcanic deermouse Peromyscus hylocetes Merriam 1898 (Cricetidae) from Parque Nacional Nevado de Colima “El Floripondio”, Jalisco, Mexico. The new species can be distinguished morphologically from the other 18 congeneric species of Moniliformis by a combination of morphological and molecular characters including the number of hooks on the proboscis (12 longitudinal rows, each one with six to eight transversally arranged unrooted hooks), the proboscis length (230–270 μm), the female trunk length (159–186 mm) and egg size (40–70 × 20–40). For molecular distinction, nearly complete sequences of the small subunit (SSU) and large subunit (LSU) of the nuclear ribosomal DNA and cytochrome oxidase subunit 1 (cox 1) of the mitochondrial DNA of the new species were obtained and compared with available sequences downloaded from GenBank. Phylogenetic analyses inferred with the three molecular markers consistently showed that Moniliformis ibunami n. sp. is sister to other congeneric species of Moniliformis. The genetic distance with cox 1 gene among Moniliformis ibunami n. sp., M. saudi, M. cryptosaudi, M. kalahariensis, M. necromysi and M. moniliformis ranged from 20 to 27%. Morphological evidence and high genetic distance, plus the phylogenetic analyses, indicate that acanthocephalans collected from the intestines of transvolcanic deer mice represent a new species which constitutes the seventh species of the genus Moniliformis in the Americas.     

Heteroplasmy and Ancient Translocation of Mitochondrial DNA to the Nucleus in the Chinese Horseshoe Bat (Rhinolophus sinicus) Complex

The utility and reliability of mitochondrial DNA sequences in phylogenetic and phylogeographic studies may be compromised by widespread and undetected nuclear mitochondrial copies (numts) as well as heteroplasmy within individuals. Both numts and heteroplasmy are likely to be common across diverse taxa yet few studies have characterised their frequencies and variation at the intra-specific level. Here we report the presence of both numts and heteroplasmy in the mitochondrial control region of the Chinese horseshoe bat Rhinolophus sinicus. In total we generated 123 sequences from 18 bats, which contained two different numt clades (i.e. Numt-1 and Numt-2) and one mtDNA clade. The sequence divergence between Numt-1 and Numt-2 was 16.8% and each numt type was found in all four R. sinicus taxa, suggesting either two ancient translocations of mitochondrial DNA into the nucleus from the same source taxon, or a single translocation from different source taxa that occurred before the split of R. sinicus into different lineages. Within the mtDNA clade, phylogenetic relationships among the four taxa of R. sinicus were similar to those seen in previous results. Based on PCR comparisons, heteroplasmy was inferred between almost all individuals of R. sinicus with respect to sequence variation. Consistent with introgression of mtDNA between Central sinicus and septentrionalis, individuals from these two taxa exhibited similar signatures of repeated sequences in the control region. Our study highlights the importance of testing for the presence of numts and heteroplasmy when applying mtDNA markers to phylogenetic studies.     

Rare hydroperoxyl guaianolide sesquiterpenes from Pulicaria undulata

Pulicaria species, such as Pulicaria undulata, are rich in sesquiterpene lactones. The methylene chloride/methanol (1:1) extract of P. undulata resulted in the isolation of new sesquiterpenes (1–4), as well as previously reported metabolites (5–14). Structures were elucidated by spectroscopic analyses. Using a mouse peritoneal macrophage bioassay, lipopolysaccharide-induced nitric oxide inhibition was observed with the eudesman-type sesquiterpene 1β,4β-dihydroxy-5αH,7αH,8α-guaia-10(14),11(13)-dien-8β,12-olide (11) at an EC₅₀ of 7.2 μM.     

One or three species in Megadenia (Brassicaceae): insight from molecular studies

Megadenia Maxim. is a small genus of the Brassicaceae endemic to East Asia with three disjunct areas of distribution: the eastern edge of the Qinghai–Tibetan Plateau, the Eastern Sayan Mountains in southern Siberia, and Chandalaz Ridge in the southern Sikhote-Alin Mountains. Although distinct species (M. pygmaea Maxim., M. bardunovii Popov, and M. speluncarum Vorob., Vorosch. and Gorovoj) have been described from each area, they have lately been reduced to synonymy with M. pygmaea due to high morphological similarity. Here, we present the first molecular study of Megadenia. Using the sequences of 11 noncoding regions from the cytoplasmic (chloroplast and mitochondrial) and nuclear genomes, we assessed divergence within the genus and explored the relationships between Megadenia and Biscutella L. Although M. bardunovii, M. speluncarum, and M. pygmaea were found to be indiscernible with regard to the nuclear and mitochondrial markers studied, our data on the plastid genome revealed their distinctness and a clear subdivision of the genus into three lineages matching the three described species. All of the phylogenetic analyses of the chloroplast DNA sequences provide strong support for the inclusion of Megadenia and Biscutella in the tribe Biscutelleae. A dating analysis shows that the genus Megadenia is of Miocene origin and diversification within the genus, which has led to the three extant lineages, most likely occurred during the Early–Middle Pleistocene, in agreement with the vicariance pattern. Given the present-day distribution, differences in habitat preferences and in some anatomical traits, and lack of a direct genealogical relationship, M. pygmaea, M. bardunovii, and M. speluncarum should be treated as distinct species or at least subspecies.