Complete sequence and genetic features of the mitochondrial genome of Pyropia tenera (Rhodophyta)

We determined the complete mitochondrial genome (mtDNA, mitogenome) of Pyropia tenera (Bangiales, Rhodophyta). Pyropia tenera mtDNA had a larger size (42,268 bp) than the mtDNA sequences of Porphyra and Pyropia reported previously, and encoded two ribosomal RNA genes [large subunit (rnl), small subunit (rns)], 24 transfer RNAs, four ribosomal proteins, and 17 genes involved in electron transport and oxidative phosphorylation. Moreover, four conserved open reading frames (ORFs) and six intronic ORFs (three in rnl and three in the cox1 gene) were also identified. In comparison with other Porphyra and Pyropia species, Py. tenera had four major structural changes in two gene loss/rearrangement regions [tRNA-Gln(uug)–tRNA-SeC(uca) and tRNA-Ala(ugc)–tRNA-Arg(ucu)] and two different patterns of exon, intron, and intronic ORFs (rnl and cox1). The unique features of Py. tenera mtDNA include the complete sequence of red algal mtDNA for investigating mtDNA evolution and developing molecular markers for species identification. In addition, red algal mtDNA can provide useful genetic information as a genetic reservoir for bioengineering.     

Use of PCR-RFLP for the discrimination of Japanese Porphyra and Pyropia species (Bangiales, Rhodophyta)

In order to distinguish 18 Porphyra and Pyropia species, the present study employed polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis using mitochondrial DNA related to the ATP synthase F0 subunit 6 (ATP6) gene and partial mitochondrial DNA including trnC, rps11, sdh3, trnG, trnN, trnP, and rns. The two primer sets on the mitochondrial DNA used in this study were able to amplify single fragments with PCR in 16 Japanese and 2 non-Japanese Porphyra and Pyropia species. Lengths of partial mitochondrial DNA related to ATP6 gene and trnC–rns ranged from 664 bp (Py. dentata and Py. haitanensis) to 677 bp (Py. lacerata and Py. kurogii) and from 1,292 bp (Py. seriata) to 1,343 bp (Py. kurogii and Py. moriensis), respectively. All 18 species were successfully distinguished using a combination of five restriction enzymes (TaqI, SspI, AciI, Cfr13I, and AluI). It was therefore concluded that PCR-RFLP analysis is a valuable tool for discrimination of wild strains of Porphyra and Pyropia species for potential use in mariculture.     

New species of unicellular obligate parasite, <Emphasis Type="Italic">Olpidiopsis pyropiae</Emphasis> sp. nov., that plagues <Emphasis Type="Italic">Pyropia</Emphasis> sea farms in Korea

Pyropia (Porphyra) sea farms are plagued with many diseases, similar to a land crop field. Olpidiopsis disease has been one of the most serious diseases causing multimillion dollars of economic loss every year. From 3 years of epidemiological studies in Pyropia sea farms, we found that the pathogen of Olpidiopsis disease in Korea is different from the oomycete, Olpidiopsis porphyrae, which is known to infect the commercially cultivated Pyropia yezoensis in Japan. The Korean species showed a clearly different small subunit (SSU) 18S rRNA sequence (90.4 % identity) and lacked four intron-like insertions, which are present in O. porphyrae. We therefore established a new species named Olpidiopsis pyropiae. The infection process and asexual life history of O. pyropiae were similar to O. porphyrae. Infection started when zoospores attached to the surface of Pyropia blade, lost flagella, and produced thin germ tubes that penetrated the cell walls of the host. Spherical multinucleate thalli developed within the cytoplasm of its algal host, which grew into mature sporangia within the next 2 days. The shape and size of sporangium was similar to that of the Japanese species, except for longer discharge tubes in O. pyropiae. Transmission electron microscopy revealed the presence of K-body-like organelles with tubular inclusions located close to the nucleus, which is one of the key characters of the genus. However, phylogenetic analysis based on SSU ribosomal RNA (rRNA) gene data showed a loose affinity of the Korean species with the other marine Olpidiopsis spp.     

Isolation and evaluation of a strain of Pyropia yezoensis (Bangiales,Rhodophyta) resistant to red rot disease

Red rot disease (Pythium porphyrae) resistant strains of Pyropia yezoensis, AP1 and AP2, were isolated from live cells taken from lesions on infected P. yezoensis blades. The degree of resistance of these strains to red rot disease was evaluated over a range of environmental conditions including temperature (10, 15, and 20 °C), salinity (20, 26, and 32 ppt), and pH (7.5, 8.0, and 8.5). These conditions are within the range that red rot disease naturally occurs on Pyropia blades. P. yezoensis and Pyropia suborbiculata with low and high partial resistance to red rot disease, respectively, were used as controls. Infection with red rot disease occurred under all environmental conditions. The incidence, severity, and expansion of the disease increased with increasing temperature and decreasing salinity and pH. The resistance of the strains P. yezoensis-AP1 and P. yezoensis-AP2 was higher than that of P. yezoensis, but lower than that of P. suborbiculata. The degree of resistance was not significantly different between the AP1 and AP2 strains. These strains can therefore be considered to exhibit stable partial resistance to P. porphyrae, and as a good starting point for the development of more resistant strains that will prevent or reduce the incidence of red rot disease on Pyropia farms.     

Cryptic species in the Pyropia yezoensis complex (Bangiales,Rhodophyta): Sympatric occurrence of two cryptic species even on same rocks

In a previous study on wild populations of Pyropia, the occurrence of two possible new species (Pyropia sp. 2 and Pyropia sp. 3) which are closely related to the two commercially important Pyropia species, P. yezoensis and P. tenera, was confirmed as the result of molecular phylogenetic analyses. To characterize the morphological features of the two wild Pyropia species, we collected Pyropia blades in a natural population in which Pyropia sp. 3 was known to occur, and carried out molecular identification before detailed morphological observations. Through the molecular identification we found, unexpectedly, that Pyropia sp. 2 blades grew sympatrically in the same site. Therefore, after molecular identification, we examined in detail the external morphology and anatomy of the two wild Pyropia species using more than 10 blades each. As a result, it is concluded that all of the blades of the two species are morphologically identical to P. yezoensis, but distinct from P. tenera. It is therefore considered that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex. Furthermore, this study revealed that the two cryptic species grew sympatrically, even on the same rocks within the natural habitat.     

Two Sympatric Phylogroups of the Chinese Water Deer (Hydropotes inermis) Identified by Mitochondrial DNA Control Region and Cytochrome b Gene Analyses

Mitochondrial DNA (mtDNA) control region (927 bp) and cytochrome b gene (1,140 bp) sequences of the Chinese water deer (Hydropotes inermis) from China and Korea were obtained to examine the taxonomic status of two subspecies, H. i. inermis from China and H. i. argyropus from Korea. Two sympatric mtDNA clades (a major clade from China and Korea and a minor clade from Korea) with an average genetic distance of 2.1% in the control region and 1.3% in the cytochrome b gene were detected. These findings are not consistent with the current classification by pelage color. We propose a reconsideration of the validity of the subspecies designation by the statistical comparison of morphological characters including body color. The major common mtDNA phylogroup in the two allopatric subspecies could be explained by the contiguous distribution of the Chinese water deer from east China to Korea until recent years. The restriction in the range and number of the Chinese subspecies after the last glacier might have caused the disappearance of the minor phylogroup in China. The taxonomic status of the two groups in Korea should be clarified using nuclear DNA marker analyses as well as morphological characters including pelage color.     

Use of the VNTR typing technique to determine the origin of Mycobacterium tuberculosis strains isolated from Filipino patients in Korea

With increasing international interchange of personnel, international monitoring is necessary to decrease tuberculosis incidence in the world. This study aims to develop a new tool to determine origin of Mycobacterium tuberculosis strains isolated from Filipino patients living in Korea. Thirty-two variable number tandem repeat (VNTR) loci were used for discrimination of 50 Filipino M. tuberculosis strains isolated in the Philippines, 317 Korean strains isolated in Korea, and 8 Filipino strains isolated in Korea. We found that the VNTR loci 0580, 0960, 2531, 2687, 2996, 0802, 2461, 2163a, 4052, 0424, 1955, 2074, 2347, 2401, 3171, 3690, 2372, 3232, and 4156 had different mode among copy numbers or exclusively distinct copy number in VNTR typing between Filipino and Korean M. tuberculosis strains. When these differences of the VNTR loci were applied to 8 Filipino M. tuberculosis strains isolated in Korea, 6 of them revealed Filipino type while 2 of them had Korean type. Using the differences of mode or repeated number of VNTR loci were very useful in distinguishing the Filipino strain from Korean strain.     

Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp.

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonas reinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropia tenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropia tenera/Chlamydomonas reinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.     

Speciation in the marine crop Pyropia yezoensis (Bangiales,Rhodophyta)

In the marine red alga Pyropia yezoensis, commonly known in Japan as nori, sympatric occurrence of two cryptic species Pyropia sp. 2 and Pyropia sp. 3 on the same rock in a natural habitat has been confirmed by molecular analysis and detailed morphological observations. To confirm whether Pyropia sp. 2 and Pyropia sp. 3 were reproductively isolated in the sympatric population, 170 blades that had previously been studied using a maternally inherited plastid marker were examined with a nuclear gene marker. The results suggested that Pyropia sp. 2 and Pyropia sp. 3 with identical morphological features were reproductively isolated in the sympatric population and that they were different species based on the biological species concept. Although gametophytic blades of Pyropia were usually assumed to be haploid, 18 of 170 blades possessed both of the two genotypes derived from Pyropia sp. 2 and from Pyropia sp. 3. These results inferred that allodiploid blades were generated from the interspecific hybridization between these two cryptic species. The present findings provide insights for future studies on the speciation mechanism in seaweeds, particularly for genera that contain numerous species.     

Characterization of a novel y-type high molecular weight glutenin subunit at <Emphasis Type="Italic">Glu-D1</Emphasis> locus

Glu-D1y12.K as a novel y-type subunit was found in HMW-GSs encoded at the Glu-D1 locus in the JB20, which a Korean wheat line from F₉ lines crossed by Keumkang with Glu-D1d and Chinese Spring (CS) with Glu-D1a alleles. This novel subunit shows faster electrophoretic mobility and lower molecular weight than Dy12 subunit on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result of linear ion-trap and Fourier-transform mass spectrometry (LTQ-FT-MS) based on two-dimensional electrophoresis (2-DE) showed that the Dy12.K subunit has high similarity against protein ID: P08488 (GLT3_WHEAT) as ‘Glutenin, high molecular weight subunit 12’ form UniProtKB. The gene of the Glu-1Dy12.K subunit is composed of 1962 nucleotide base pairs containing open reading frame (ORF) as 652 amino acids corresponding to about 70.1 kDa. It has four indels (36 bp insertions: two repeated 18 and 24 bp deletion: two deletions with 6?+?18 bp) and 21 SNPs compared to Glu-1Dy10 (GI: 164457872 in NCBI), and one deletion (18 bp) and three SNPs compared to Glu-1Dy12 (GI: 1036031968) by DNA markers. Consequentially, in comparison with Dy10, 13 SNPs were non-synonymous SNPs and eight SNPs were synonymous SNPs of 21 SNPs. In comparison with Dy12, only one SNP was non-synonymous SNP of three SNPs. Furthermore, the deduced peptide sequences as ‘TGQGQQ’ corresponding to ‘AACAGGACAAGGGCAACA’ are deleted only in the Dy12.K subunit.     

Overexpression of the Bx7 high molecular weight glutenin subunit on the <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">B1</Emphasis> locus in a Korean wheat landrace

The Glu-B1al (Bx7^OE + By8) allele is important for bread-making quality. The allele was found in a Korean wheat landrace using specific DNA markers. Molecular analyses were conducted to identify the overexpressed Bx7 (Bx7^OE) subunit of the allele. The Korean wheat landrace (accession ID: IT166460) showed a similar protein expression level of Bx7 subunit, i.e., overexpression of Bx7 subunit towards cv. Glenlea, Canadian Western Red Spring wheat, which harbors Bx7^OE subunit of Glu-B1al as detected on SDS–PAGE (sodium dodecyl sulfate poly-acrylamide gel electrophoresis). In addition, 2-DE (two-dimensional electrophoresis) analysis revealed similar protein expression patterns of the Bx7 subunit regions of IT166460 and Glenlea. The proportion of Bx7 to total HMW-GSs (high molecular weight glutenin subunits) in IT166460 (56.17 ± 0.22%) was higher than that of Chinese Spring (34.75 ± 1.03%) and even that of Glenlea (46.25 ± 1.76%) as assessed by RP-HPLC (reverse-phase high-performance liquid chromatography). Overexpression of Bx7 subunit was caused by gene duplication and indels of the promoter region of the Bx7 gene. IT166460 attained the 43 bp indel of the promoter region, as did Glenlea, i.e., the amplicon size of IT166460 was the same as that of Glenlea. In addition, the nucleotides present in the duplicated gene in IT166460 were the same as those in Glenlea. Bx7^OE subunit is critical for dough strength. However, most wheat accessions harboring the subunit are distributed in America. Furthermore, most Korean wheats have little genetic variation in glutenin composition and are associated with inferior bread quality. Hence, IT166460 could be used to improve bread-making quality in the Korean wheat breeding program.     

Different types of fruit damages of three internal apple feeders diagnosed with mitochondrial molecular markers

Three tortricid pests, Grapholita dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura), are known as internal apple feeders in Korea. To identify young larvae, this study developed two types of molecular markers from their mitochondrial DNA (mtDNA) sequences. To this end, six different loci of mtDNA were sequenced in G. dimorpha: cytochrome oxidase subunit I (460 bp), cytochrome oxidase subunit II (446 bp), cytochrome b (308 bp), NADH dehydrogenase 3 (585 bp), NADH dehydrogenase 4 (ND4, 835 bp), and 16S rRNA (1300 bp). These sequences were compared with those of G. molesta and C. sasakii in order to develop PCR–RFLP and diagnostic primers. ND4 locus was selected to be used for developing a PCR–RFLP marker. ND4-Swa I digests showed two bands for G. dimorpha, one band for G. molesta, and three bands for C. sasakii. On the other hand, species-specific diagnostic PCR primers were developed using ND4 locus. These markers were then applied to diagnose larvae infesting apples to determine species-specific fruit damage patterns, in which G. dimorpha, G. molesta, and C. sasakii showed different feeding behaviors in terms of their main feeding sites in apple fruits.     

Molecular profiling of a y-type high molecular weight glutenin subunit at <Emphasis Type="Italic">Glu-D1</Emphasis> locus from a North Korean landrace wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The objective of this study is to demonstrate characteristics of a y-type high molecular weight glutenin subunit (D1y HMW-GS) at Glu-D1 found in IT212991, a North Korean landrace wheat compared to Dy12 and Dy12.K as a novel HMW-GS in JB20, a Korean wheat line onto molecular analyses as PCR, cloning, DNA sequencing, and RP-HPLC and proteomic analyses as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), two-dimensional electrophoresis (2-DE), Fourier-transform mass spectrometry (LTQ-FT-MS). The D1y of IT212991 was identified to have faster electrophoretic mobility than that of Dy12 by SDS–PAGE. HMW-GS components of IT212991 were identified to be different from Chinese Spring (CS) and JB20, a Korean wheat line by RP-HPLC. The result of mass spectrometric analysis, the D1y of IT212991 (68510.8 Da) was similar to that of Dy12.K of JB20 (68514.4 Da), and lower than Dy12 of CS (69151.2 Da). The result of LTQ-FT-MS based on 2-DE, the D1y of IT212991 was identified to be similar with Dy12 corresponding to the protein function as ‘Glutenin, high molecular weight subunit 12’. The D1y encoding the D1y of IT212991 was identified to consist of 652 amino acid sequences corresponding to 1962 bp according to DNA sequencing. The gene was identified to have a insertion and deletion (InDel) corresponding to 18 bp sequences ‘AACAGGACAAGGGCAACA’ compared to ordinary Dy12 gene. It was demonstrated that the D1y of IT212991 is the same as Dy12.K.     

Development of SSR markers by next-generation sequencing of Korean landraces of chamoe (Cucumis melo var. makuwa)

The oriental melon (Cucumis melo var. makuwa), called ‘chamoe’ in Korean, is a popular fruit crop cultivated mainly in Asia and a high-market value crop in Korea. To provide molecular breeding resources for chamoe, we developed and characterized genomic SSR markers from the preliminary Illumina read assemblies of Gotgam chamoe (one of the major landraces; KM) and SW3 (the breeding parent). Mononucleotide motifs were the most abundant type of markers, followed by di-, tri-, tetra-, and pentanucleotide motifs. The most abundant dinucleotide was AT, followed by AG and AC, and AAT was the most abundant trinucleotide motif in both assemblies. Following our SSR-marker development strategy, we designed a total of 370 primer sets. Of these, 236 primer sets were tested, exhibiting 93 % polymorphism between KM and SW3. Those polymorphic SSRs were successfully amplified in the netted and Kirkagac melons, which respectively exhibited 81 and 76 % polymorphism relative to KM, and 32 and 38 % polymorphism relative to SW3. Seven selected SSR markers with a total of 17 alleles (2–3 alleles per locus) were used to distinguish between KM, SW3, and four chamoe cultivars. Our results represent the first attempt to provide genomic resources for Korean landraces for the purposes of chamoe breeding, as well as to discover a set of SSR markers capable of discriminating chamoe varieties from Korea and the rest of Asia, which possess little genetic diversity. This study establishes a highly efficient strategy for developing SSR markers from preliminary Illumina assemblies of AT-rich genomes.     

The complete mitochondrial genome of the Korean red-backed vole, Myodes regulus (Rodentia, Murinae) from Korea

We have determined the complete mitochondrial genome (NC_016427) of the Korean red-backed vole, Myodes regulus, which is distributed in South Korea. The total length of the M. regulus mitogenome is 16,379?bp, with a base composition of 33.0% A, 26.7% T, 27.1% C, and 13.2% G. The total length of the 13 protein-coding genes is 11,396?bp long.     

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

Nitrogen biofiltration capacities and photosynthetic activity of Pyropia yezoensis Ueda (Bangiales,Rhodophyta): groundwork to validate its potential in integrated multi-trophic aquaculture (IMTA)

Porphyra spp. (currently Porphyra and Pyropia) are major sources of seafood globally. In this study, we investigated the effects of ammonium concentration, water temperature, and thallus stocking density on N-ammonium uptake rate (NUR), tissue nutrients content, N–NH₄ ⁺ filtration efficiency (NUE: nitrogen uptake efficiency %) of Pyropia yezoensis at a laboratory scale and in a mesoscale to evaluate the potential of this species as a biofilter. Additionally, photosynthetic activity was examined using Diving-PAM fluorometer to evaluate the health status. At a laboratory scale, the NUR and tissue nitrogen (N) content of P. yezoensis increased with increasing NH₄ ⁺ concentrations in the medium. The NUR at thallus stocking densities of 5 and 10 g fresh weight (FW) L^–1 were significantly higher than that at 20 g FW L^–1. Effective quantum yield (? F/F’ _m ) and tissue N content was significantly higher at all stocking densities than that at the beginning of experiment. The NUE was over 90 % at 10 and 17 °C, while all thalli cultured at 25 °C died after 5 days. In a mesoscale, the NUE at a thallus stocking density of 10.0 g FW L^–1 was significantly higher than that at a stocking density of 5.0 g FW L^–1. No differences in the NUE occurred between 10 °C and 17 °C. Photosynthetic activity (?F/F’_m and rETR_max) of P. yezoensis at optimal culture condition (10–12 °C and 10 g FW L^–1) increased over time through the experiment. This indicates that thallus was healthy during culture and chlorophyll a fluorescence can be as a monitoring tool for evaluating the physiological status of seaweeds in an integrated multi-trophic aquaculture.     

Genetic diversity of genus Vespa including an invaded species of V. velutina (Hymenoptera: Vespidae) in Korea inferred from DNA barcoding data

With about 5000 known species, the Vespidae is a large family belongs to order Hymenoptera. The genus Vespa with 22 species is one of the four genera of the subfamily Vespinae. In Korea, 10 species and subspecies are recognized. Because of their social behavior, their treat to human health and their impact in apiculture, the reliable and sometimes automated identification of these insects to species level are important. To test the efficacy of DNA barcoding method for identification of species of the genus Vespa in Korea, 30 samples of eight Korean species of genus Vespa were collected and mitochondrial DNAs of 658 bp fragment cytochrome oxidase subunit 1 (CO1) region were sequenced. A Bayesian Inference based on COI gene of the Korean Vespa species was constructed. The phylogenetic tree shoed that identification of all specimens is possible based on COI gene and we found strong relation between the sequences of the collected species from different localities in South Korea which clustered together with 100% support with sequences of the same species in GenBank. The results demonstrate that DNA barcoding is a useful technique for rapid and accurate species recognition in Korean Vespa species. The DNA barcode part of COI for V. binghami is provided for the first time that can help for identification of this species through DNA barcoding. Also, the genetic diversity among Korean Vespa velutina was zero suggests that the invasion might have occurred in a single event with small number of founders.