Molecular phylogeny of mangroves V. Analysis of genome relationships in mangrove species using RAPD and RFLP markers

 DNA from pooled leaf samples of 11 true major mangrove, three true minor mangrove, two mangrove associate, two mangrove parasite, three terrestrial and one cultivated species were isolated for the present study. In total, 198 random amplified polymorphic DNAs (RAPDs) and 180 restriction fragment length polymorphism (RFLP) loci were scored by using ten primers and 14 enzyme-probe combinations respectively. The polymorphism observed for these markers revealed a high degree of genetic diversity in mangroves at both inter-specific or inter-generic levels. A dendrogram, constructed after pooling both RAPD and RFLP data, using a similarity index was analysed for genome relationships among these species. The dendrogram showed clustering of all the major mangroves, except for Nypa fruticans (Arecaceae), into one group. All species under the tribe Rhizophorae formed a sub-cluster, to which Xylocarpus granatum was found to be the most closesly related species. The clustering pattern implied that Excoecaria agallocha and Acanthus ilicifolius should be considered as true minor mangroves. The present study also provided molecular data favouring the separation of Avicennia spp. from the Verbenaceae to create a monotypic family the Avicenniaceae. The separation of Viscum orientale into the Viscaceae was also favoured. Received: 5 March 1998 / Accepted: 1 April 1998     

Molecular phylogeny of mangroves VII. PCR-RFLP of trnS-psbC and rbcL gene regions in 24 mangrove and mangrove-associate species

Chloroplast DNA (cpDNA) regions, trnS-psbC and rbcL, from 120 individuals of 24 mangrove and mangrove associate species belonging to 11 orders, 13 families and 17 genera of Angiospermae were amplified by the polymerase chain reaction (PCR) and restriction-digested with HaeIII. Analysis of polymorphism in the restriction fragments (PCR-RFLP) revealed 18 classes of restriction banding pattern in trnS-psbC region. This has provided molecular evidence for diversity in the mangrove floral component at the above-species level. Intra-generic variations were observed in three genera, viz. Rhizophora, Avicennia and Suaeda. Species-specific restriction patterns were found in the genera Rhizophora and Suaeda. A natural hybrid belonging to the genus Rhizophora was also analysed, and its restriction pattern was the same as that of a putative parental species.PCR-RFLP analysis of rbcL gene region was less differentiating. However, it showed 13 different classes of restriction patterns and revealed the usefulness of these investigations for genome analysis at a higher taxonomic level. Intra-specific variation was not observed in any of the species in either of the cpDNA regions analysed. This is the first report which describes variations in the chloroplast genome of mangrove species. Received: 20 April 1999 / Accepted: 12 May 1999     

Molecular phylogeny of gibbons inferred from mitochondrial DNA sequences: Preliminary report

We analyzed the 896 base-pair (bp) mitochondrial DNA (mtDNA) sequences for seven gibbons, representative of three out of four subgenera. The result from our molecular analysis is consistent with previous studies as to the monophyly of subgenus Hylobates species, yet the relationship among subgenera remains slightly ambiguous. A striking result of the analysis is the phylogenetic location of Kloss's gibbon (H. klossii). Kloss's gibbon has been considered to be an initial off-shoot of the subgenus Hylobates because of its morphological primitiveness. However, our molecular data strongly suggest that Kloss's gibbon speciated most recently within the subgenus Hylobates. Correspondence to: S. Horai     

Molecular phylogeny of the Chinese ranids inferred from nuclear and mitochondrial DNA sequences

The phylogeny of representative species of Chinese ranids was reconstructed using two nuclear (tyrosinase and rhodopsin) and two mitochondrial (12S rRNA, 16S rRNA) DNA fragments. Maximum parsimony, Bayesian, and maximum likelihood analyses were employed. In comparison with the results from nuclear and mitochondrial data, we used nuclear gene data as our preferred phylogenetic hypothesis. We proposed two families (Ranidae, Dicroglossidae) for Chinese ranids, with the exception of genus Ingerana. Within Dicroglossidae, four tribes were supported including Dicroglossini, Paini, Limnonectini, and Occidozygini. A broader sampling strategy and evidence from additional molecular markers are required to decisively evaluate the evolutionary history of Chinese ranids.     

Molecular phylogeny of the subfamily Schizothoracinae (Teleostei: Cypriniformes: Cyprinidae) inferred from complete mitochondrial genomes

The schizothoracine fishes, members of the Teleost order Cypriniformes, are one of the most diverse group of cyprinids in the Qinghai–Tibetan Plateau and surrounding regions. However, taxonomy and phylogeny of these species remain unclear. In this study, we determined the complete mitochondrial genome of Schizopygopsis malacanthus. We also used the newly obtained sequence, together with 31 published schizothoracine mitochondrial genomes that represent eight schizothoracine genera and six outgroup taxa to reconstruct the phylogenetic relationships of the subfamily Schizothoracinae by different partitioned maximum likelihood and partitioned Bayesian inference at nucleotide and amino acid levels. The schizothoracine fishes sampled form a strongly supported monophyletic group that is the sister taxon to Barbus barbus. A sister group relationship between the primitive schizothoracine group and the specialized schizothoracine group + the highly specialized schizothoracine group was supported. Moreover, members of the specialized schizothoracine group and the genera Schizothorax, Schizopygopsis, and Gymnocypris were found to be paraphyletic.     

Phylogenetic relationships inCrossostylis (Rhizophoraceae) inferred from restriction site variation of chloroplast DNA

Phylogenetic relationships among the nine species ofCrossostylis (Rhizophoraceae) were elucidated using cladistic analysis of restriction site variations of chloroplast DNA. As a result, this genus was found to comprise two pronounced monophyletic groups as follows:C. biflora, C. grandiflora, C. multiflora andC. sebertii; andC. cominsii, C. pachyantha, C. parksii, C. richii andC. seemannii. Moreover, the monophyly ofC. biflora, C. grandiflora andC. sebertii in the former group and the monophyly ofC. pachyantha, C. parksii, C. richii andC. seemannii in the latter group were also suggested. The molecular tree corresponded well with that inferred from morphological data and no discrepancy was recognized. Many of the floral morphological characters reflected lineage, but all seed coat characters were homoplasious. Evolutionary trends in some morphological characters were optimized on the cpDNA tree obtained. Species from New Caledonia and Polynesia were monophyletic, as were those from the Solomon Islands, Vanuatu and the Fiji Islands. All species endemic to the Fiji Islands made a cluster, and this suggests that speciation occurred from a single ancestral species on the Islands.     

尼罗鳄线粒体基因组全序列分析及鳄类系统发生关系的探讨

大多数脊椎动物的线粒体基因组（约16—18kb）的组成是相对较稳定的,但在不同类群中,线粒体基因组在基因结构和基因排列方式等方面均显示了极大的多样性,这种多样性可能反映了真核细胞不同的进化路线（Saccone et al．,1999）。就目前的研究而言,线粒体基因组是惟一一个能够从基因组水平上来分析动物系统发生的分子标记,可以从线粒体基因组序列信息、基因组成及基因排列方式等进行多方位的分子进化研究,因而线粒体基因组全序列将成为动物分子系统发生最有力的证据（Saccone et al．,1999）。     

Molecular phylogeny of the family Dicroglossidae (Amphibia: Anura) inferred from complete mitochondrial genomes

The fork-tongued frogs, members of the amphibian Order Anura, belong to the family Dicroglossidae and are one of the most diverse groups of Anuran frogs; however, their taxonomy and phylogeny remain controversial. In the present study, sixteen dicroglossine mitochondrial genomes representing nine dicroglossine genera and 23 other neobatrachian taxa, were used to reconstruct the phylogenetic relationships of the family Dicroglossidae using different partitioned maximum likelihood and partitioned Bayesian inference methods at both the nucleotide and amino acid levels. The sampled fork-tongued frogs form a strongly supported monophyletic group that is the sister taxon to another well-supported clade that includes representatives of the families Ranidae, Rhacophoridae, and Mantellidae. The monophyly of the subfamily Occidozyginae and Dicroglossinae was revealed with strong supports, and two major clades were supported within Dicroglossinae. The sister-group relationship between the genera Limnonectes and the tribe Paini was supported. In addition, a sister-group relationships between Fejervarya and Euphlyctis + Hoplobatrachus, between Quasipaa and Yerana, and between Feirana and Nanorana are well supported. Estimates of divergence times revealed the divergence of Dicroglossidae during the Late Upper Cretaceous to the Early Eocene, and diversification of the major dicroglossine genera from the Early Eocene to the Middle Miocene.     

A phylogeny of Chinese species in the genus Phrynocephalus (Agamidae) inferred from mitochondrial DNA sequences

We investigated the phylogenetic relationships among most Chinese species of lizards in the genus Phrynocephalus (118 individuals collected from 56 populations of 14 well-defined species and several unidentified specimens) using four mitochondrial gene fragments (12S rRNA, 16S rRNA, cytochrome b, and ND4-tRNA(LEU)). The partition-homogeneity tests indicated that the combined dataset was homogeneous, and maximum-parsimony (MP), neighbor-joining (NJ), maximum-likelihood (ML) and Bayesian (BI) analyses were performed on this combined dataset (49 haplotypes including outgroups for 2058bp in total). The maximum-parsimony analysis resulted in 24 equally parsimonious trees, and their strict consensus tree shows that there are two major clades representing the Chinese Phrynocephalus species: the viviparous group (Clade A) and the oviparous group (Clade B). The trees derived from Bayesian, ML, and NJ analyses were topologically identical to the MP analysis except for the position of P. mystaceus. All analyses left the nodes for the oviparous group, the most basal clade within the oviparous group, and P. mystaceus unresolved. The phylogenies further suggest that the monophyly of the viviparous species may have resulted from vicariance, while recent dispersal may have been important in generating the pattern of variation among the oviparous species.     

Molecular phylogeny of mangroves I. Use of molecular markers in assessing the intraspecific genetic variability in the mangrove species Acanthus ilicifolius Linn. (Acanthaceae)

 Mangroves, the intertidal ecosystems occurring primarily in the tropical regions of the world, are valuable natural resources with high productivity and unique habitat value. However, the genetic structure of plant species within the mangrove ecosystem is poorly understood. The present communication is the first report on the use of molecular markers in assessing intra-site and intra-specific polymorphism in one of the mangrove species, Acanthus ilicifolius, for identifying/ detecting distinct genotypes for long-term conservation. Random amplified polymorphic DNAs (RAPDs) and restriction fragment length polymorphisms (RFLPs) were used to elucidate the intra- and inter-population variability in this widely distributed mangrove species. In all, 48 genotypes representing eight distinct populations were analysed. A low level of polymorphism was detected at the intra-population level through both RAPD (3.8–7.3%) and RFLP (3.2–9.1%) analyses. At the inter-population level, 25 of the 73 RAPD loci (34%) detected through the use of 13 random primers and 44 of the 96 RFLP loci (45.8%) revealed through 15 probe/enzyme combinations were polymorphic. RFLP analyses were carried out using genomic clones developed from the same species. The somatic cells of the species displayed 48 chromosomes, with no numerical changes at either intra- or inter- population levels. Received: 5 June 1996 / Accepted: 8 November 1996     

Molecular phylogeny of mangroves. VI. Intraspecific genetic variation in mangrove species Excoecaria agallocha L. (Euphorbiaceae).

Genomic DNA from 84 individuals of Excoecaria agallocha from seven mangrove populations were analysed for random amplified polymorphic DNAs (RAPDs) using 16 random 10-mer primers. Polymorphism within populations varied from 20% to 31%. At the interpopulation level, 111/149 (74%) of RAPDs were polymorphic. Restriction fragment length polymorphism (RFLP) analysis of 21 individuals (3 individuals randomly selected from the 7 populations) using 30 probe-enzyme combinations revealed a high level of interpopulation polymorphism (62.2%) indicating interpopulation genetic divergence. The polymorphic RAPDs and RFLPs were pooled, and clustering was carried out based on mean similarity for individual populations. The dendrogram showed groupings of populations from the West and East Coasts of India into separate clusters, at 60% similarity level. Further, RAPD and RFLP analysis of male and female plants showed approximately the same level of variation in both sexes, and no sex-linked markers were found. These results demonstrate that considerable intrapopulation and interpopulation genetic variations exist in E. agallocha, and that lack of genetic variation is not the reason for the morphological uniformity observed across the range of the species.     

Reproductive biology and population genetic structure of Kandelia candel (Rhizophoraceae), a viviparous mangrove species

The pollination biology, mating system, and population genetic structure of Kandelia candel were investigated. Field observations on its pollination and reproductive biology suggested that this species is pollinator dependent for fruit set, and bee activities can lead to substantial geitonogamous selfing. Quantitative analysis of the mating system parameters was performed using progeny arrays assayed for allozyme markers. Multilocus outcrossing rates (t(m)) were estimated to be 0.697 ± 0.091 and 0.797 ± 0.062 in two populations. In comparison to other plant species with mixed-mating system, the level of allozyme variation was very low in the 13 populations sampled along the coastlines of Hong Kong. At the species level, the proportion of polymorphic loci was 20%, number of alleles per locus was 1.2, and heterozygosity was 0.0362. The total gene diversity was primarily distributed within populations (H(S )= 0.0339), and the coefficient of genetic differentiation among populations was low (G(ST )= 0.064). This pattern of population genetic structure suggests that gene flow, primarily in the form of water-dispersed seedlings in viviparous mangrove species, is not as limited as previously thought. However, microgeographic pattern in allele frequency at the marker loci could still be detected between the western and eastern coastal populations.     

Molecular phylogeny of Gossypium species by DNA fingerprinting

Total genomic DNA from 31 available Gossypium species, three subspecies and one interspecific hybrid, were analysed to evaluate genetic diversity by RAPD, using 45 random decamer primers. A total of 579 amplified bands were observed, with 12.9 bands per primer, of which 99.8% were polymorphic. OPJ-17 produced the maximum number of fragments while the minimum number of fragments was produced with primer OPA-08. Cluster analysis by the unweighted paired group method of arithmetic means (UPGMA) showed six main clusters. Cluster ’A’ consisted of two species and one subspecies of the A-genome, with a 0.78–0.92 Nei’s similarity range. Cluster B, composed of all available tetraploid species and one interspecific hybrid, showed the same sister cluster. Nei’s similarity ranged from 0.69 to 0.84. The B-genome formed the UPGMA sister cluster to the E-genome species. Cluster ’C’ consisted of five Gossypium species of which three belong to the B-genome, with Nei’s similarity values of 0.81 to 0.86. Although there was considerable disagreement at lower infra-generic ranks, particularly among the D- genome (diploid New World species) and C-genome (diploid Australian species) species. The sole F-genome species Gossypium longicalyx was resolved as a sister group to the D-genome species. Gossypium herbaceum and G. herbaceum Africanum showed the maximum Nei’s similarity (0.93). Minimum similarity (0.29) was observed between Gossypium trilobum and Gossypium nelsonii. The average similarity among all studied species was 50%. The analysis revealed that the interspecific genetic relationship of several species is related to their centre of origin. As expected, most of the species have a wide genetic base range. The results also revealed the genetic relationships of the species Gossypium hirsutum to standard cultivated Gossypium barbadense, G. herbaceum and Gossypium arboreum. These results correspond well with previous reported results. The level of variation detected in closely related genotypes by RAPD analysis indicates that it may be a more efficient marker than morphological marker, isozyme and RFLP technology for the construction of genetic linkage maps. Received: 2 January 2000 / Accepted: 12 February 2000     

Tracing plant Mitochondrial DNA evolution: rearrangements of the ancient mitochondrial gene cluster trnA-trnT-nad7 in liverwort phylogeny

Whereas frequent recombination characterizes flowering plant mitochondrial genomes, some mitochondrial gene arrangements may, in contrast, be conserved between streptophyte algae and early land plant clades (bryophytes). Here we explore the evolutionary fate of the mitochondrial gene arrangement trnA-trnT-nad7, which is conserved among the alga Chara, the moss Physcomitrella, and the liverwort Marchantia, although trnT is inverted in orientation in the latter. Surprisingly, we now find that the Chara-type gene arrangement is generally conserved in mosses, but that trnT is lacking between trnA and nad7 in all simple-thalloid and leafy (jungermanniid) liverworts. The ancient gene continuity trnA-trnT-nad7 is, however, conserved in Blasia, representing the sister lineage to all other complex-thalloid (marchantiid) liverworts. The recombinogenic insertion of short sequence stretches, including nad5 and rps7 pseudogene fragments copied from elsewhere in the liverwort mtDNA, likely mediated a subsequent inversion of trnT and flanking sequences in a basal grade of marchantiid liverworts, which was then followed by an independent secondary loss of trnT in derived marchantiid taxa later in evolution. In contrast to the previously observed extreme degree of coding sequence conservation and the assumed absence of active recombination in Marchantia mtDNA, this now reveals a surprisingly dynamic evolution of marchantiid liverwort mitochondrial genomes.     

Molecular phylogeny of the paper wasp subgenus Polistes (Polistella) Ashmead, 1904 (Hymenoptera: Vespidae: Polistinae) from Vietnam

This study provides the first molecular phylogeny of the social wasp subgenus Polistella (Hymenoptera: Vespidae: Polistes) from Vietnam. Fragments of the mitochondrial COI and 16S rRNA genes were used to reconstruct the phylogenetic trees among 38 Polistes species plus two out-group species (Vespa soror du Buysson and Ropalidia fasciata (Fabricius)). Our results support the existence of several species-groups, including two that are congruent with the previous stigma and Stenopolistes groups defined on the basis of morphology. Moreover, we recovered a clade including the stigma group and the two species P. humilis and P. variabilis that was sister to all other species of Polistella. However, the results also challenged the definition of other groups of Polistella based on morphological data, as well as the definition of two species: P. brunus and P. affinis. This first study calls for further analyses including morphological characters to clarify the taxonomy and the classification of the group.     

Nomenclatural notes and typification of names in the mangrove tribe Rhizophoreae (Rhizophoraceae) of India

The nomenclatures of the taxa of the mangrove Rhizophoraceae of India have been scrutinized and 20 names are lectotypified or neotypified. In addition, the holotypes of some names are located for the first time with clarifications provided. Since natural hybridization is common in Rhizophoraceae, its consequences for the correct application of names is also briefly discussed.     

PCR-RFLP of mitochondrial DNA reveals two origins of Apis mellifera in Taiwan

Beekeeping has been a highly valued industry in Taiwan. As a result, many subspecies of Apis mellifera have been introduced to Taiwan since 1911, leading to the hybridization of different subspecies. In order to know the matrilineal origins of Taiwan A. mellifera, a total of 280 samples collected from 33 apiaries throughout the island were examined. Using PCR-RFLP of four mitochondrial gene fragments, i.e., the non-coding region between tRNA^leu and cytochrome c oxidase subunit II (intergenic tRNA^leu-COII), cytochrome b (Cyt b), large subunit rRNA (Ls rRNA) and cytochrome c oxidase subunit I (COI), we only found two haplotypes exist in 280 samples. Haplotypes ababa and bbbaa account for 87% of these Western bees belonged to the Eastern European (C) lineage and 13% belonged to the Middle East (Z) lineage, respectively, with the latter being totally absent in northern Taiwan. African (A) and Mellifera (M) lineages, officially imported once in 1990s and 1930s respectively, were not detected. The identification of subspecies of A. mellifera and survey of their distribution on the island are expected to facilitate efficient breeding programs and establish a more booming beekeeping industry.     

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.     

Molecular phylogeny of Eurasian badgers (Meles) around the distribution boundaries,revealed by analyses of mitochondrial DNA and Y-chromosomal genes

Based on previous molecular and morphological analyses, Eurasian badgers are currently classified into Meles meles distributed in Europe, M. canescens in the Caucasus and Middle East, M. leucurus in continental Asia and M. anakuma in Japan. The precise locations of their distribution boundaries are still unclear. Therefore, in the present study, we clarified the phylogenetic relationships in the genus Meles around its distribution boundaries, based on mitochondrial DNA (mtDNA) and Y-chromosomal genes. From 71 badgers examined, 29 mtDNA haplotypes were identified, including new 26 types. Multiple haplotypes of SRY and CAN-SINEs were identified from 23 males, including two new haplotypes of SRY. The mtDNA phylogeny showed that the Ural Mountains were not a current distribution boundary between M. meles and M. leucurus. In addition, our results supported the hypothesis that the Bosphorus Strait is a geographical barrier between M. meles and M. canescens. The badgers from Bulgaria north of the Bosphorus Strait shared haplotypes with M. meles. On the other hand, badgers from Far Eastern Russia distributed in the eastern peripheral region of the Asian Continent had haplotypes of M. leucurus. The badgers from Norway and Finland shared haplotypes with M. meles.     

Molecular phylogeny and systematics of the Barbinae (Teleostei: Cyprinidae) in China inferred from mitochondrial DNA sequences

The taxonomy and phylogeny of the Chinese species of the Barbinae (Cypriniformes) has a confusing history. In this study, partial sequences of four mitochondrial genes (cyt b, COI, ND4 and 16S rRNA) from 75 Barbinae species and 38 outgroup species were used to investigate the taxonomy and phylogeny within the Barbinae in China. The monophyly of Neolissochilus, Sikukia and Tor are not supported. Neolissochilus benasi might represent a new genus, and Tor hemispinus and Tor qiaojiensis should be moved into Neolissochilus. Sikukia flavicaudata is not Sikukia species. Puntius paucimaculatus might be a synonym of Puntius semifasciolatus. Puntius semifasciolatus does not belong to Puntius. Onychostoma barbatum might consist of more than one species. Our molecular results corroborate that Acrossocheilus stenotaeniatus is a synonym of Acrossocheilus longipinnis. Finally, Barbonymus gonionotus from Menglun, Yunnan should be Poropuntius huangchuchieni.