Ji Tan

Phylogenetic relationship of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta) in Malaysia

The genera Kappaphycus Doty and Eucheuma J. Agardh are important sources of carrageenan in Malaysia, offering lucrative revenues to the carrageenan industry, economy, and the local community. The extensive range of morphotypes and the lack of distinct morphological characteristics led to the application of molecular systematics in elucidating this taxonomic confusion. Local varieties of Kappaphycus and Eucheuma, identified using putative external morphology, were analyzed using the mitochondrial cox2–3 spacer and plastid RuBisCO spacer molecular markers. Phylogenetic analysis of these and non-local specimens indicate that Kappaphycus and Eucheuma are genetically distinct. Three main genotypes of Kappaphycus alvarezii were identified, of which two are extant in Hawaii. Morphological and color variations are not supported by molecular data, indicating that most of the local names are not genetically based. Both the cox2–3 spacer and RuBisCO spacer generated phylogenetic trees with similar topology except in variation of nodal supports. The two markers showed clear separation between Kappaphycus and Eucheuma and the existence of three Malaysian Kappaphycus cultivars. Cox2–3 spacer data is more variable and provides better resolution than the RuBisCO spacer, showing that Kappaphycus is more diversified with a larger number of genotypes, strains, and species which are unique to Southeast Asia. Kappaphycus sp. “Aring-aring” appeared to be phenotypically and genotypically different from other Kappaphycus congeners, whereas Kappaphycus striatum exhibited two different genotypes. Our data indicate that Eucheuma denticulatum is the dominant species in Malaysian waters and also suggested paraphyly in Eucheuma which will require further studies. The application of molecular taxonomy on Malaysian Kappaphycus and Eucheuma proves useful, offering valuable insights into the taxonomy and distribution of these commercially important Rhodophytes.

Assessment of Four Molecular Markers as Potential DNA Barcodes for Red Algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta)

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.

Distinct Genetic Lineages of Bactrocera caudata (Insecta: Tephritidae) Revealed by COI and 16S DNA Sequences

The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.

Genetic diversity of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta) in Southeast Asia

The commercial importance of carrageenophytes Kappaphycus and Eucheuma is well known, with much interest in terms of cultivation, marketing, and research. Considering the many lucrative prospects, these red seaweeds were introduced into various parts of the world for farming, where merely a few were comprehensively documented. Despite being extensively cultivated throughout Southeast Asia, the genetic diversity of Kappaphycus and Eucheuma is poorly studied, where heavy reliance is placed on the use of local or commercial names for identifications. This study used the mitochondrial-encoded cox1 and cox2–3 spacer genetic markers to investigate the Kappaphycus and Eucheuma haplotypes, cultivated and wild, available throughout Southeast Asia. Concatenated cox1–cox2–3 spacer datasets were also analyzed. The near full-length cox1 gene is preferred at revealing the genetic diversity of Kappaphycus and Eucheuma, provided a larger reference database is available. Both molecular markers were capable of delineating common members of the genus Kappaphycus (i.e., Kappaphycus alvarezii, Kappaphycus striatus, and Kappaphycus cottonii) and Eucheuma denticulatum, and revealed interesting genotypes and new species which may be potential alternatives to the common cultivars as well as materials for research. The relative scarcity of Eucheuma species is discussed and future sites for sampling are recommended.

Kappaphycus malesianus sp. nov.: a new species of Kappaphycus (Gigartinales, Rhodophyta) from Southeast Asia

A new species, Kappaphycus malesianus, is established as a new member of the genus Kappaphycus. Locally known as the “Aring-aring” variety by farmers in Malaysia and the Philippines, this variety has been commercially cultivated, often together with Kappaphycus alvarezii due to the similarities in morphology. Despite also producing kappa-carrageenan, the lower biomass of the K. malesianus when mixed with K. alvarezii ultimately affects the carrageenan yield. Morphological observations, on both wild and cultivated plants, coupled with molecular data have shown K. malesianus to be genetically distinct from its Kappaphycus congeners. The present study describes the morphology and anatomy of this new species as supported by DNA data, with additional morphological features for distinguishing between commercial Kappaphycus cultivars.

Molecular phylogeny of Orthetrum dragonflies reveals cryptic species of Orthetrum pruinosum

Dragonflies of the genus Orthetrum are members of the suborder Anisoptera, family Libellulidae. There are species pairs whose members are not easily separated from each other by morphological characters. In the present study, the DNA nucleotide sequences of mitochondrial and nuclear genes were employed to elucidate the phylogeny and systematics of Orthetrum dragonflies. Phylogenetic analyses could not resolve the various subfamilies of the family Libellulidae unequivocally. The nuclear 28S rRNA gene is highly conserved and could not resolve congeneric species of Orthetrum. Individual mitochondrial genes (COI, COII, and 16S rRNA) and combination of these genes as well as the nuclear ITS1&2 genes clearly differentiate morphologically similar species, such as the reddish species pairs O. chrysis and O. testaceum, and the bluish-coloured species O. glaucum and O. luzonicum. This study also reveals distinct genetic lineages between O. pruinosum schneideri (occurring in Malaysia) and O. pruinosum neglectum (occurring north of Peninsular Malaysia from India to Japan), indicating these taxa are cryptic species.

Distinct genetic clades of Malaysian Copera damselflies and the phylogeny of platycnemine subfamilies.

The phylogenetic relationships of some taxa in the Platycnemidinae at the species and generic levels have been investigated. Phylogenetic trees were generated from both individual mitochondrial encoded COI, COII, 16S rDNA and nuclear encoded 28S rDNA and also combined sequences; these data indicate that the component taxa of the genus Copera belong to two distinct genetic clades – the marginipes group and the annulata group. There was no distinct genetic difference between the red-legged and yellow-legged morphs of C. vittata. Molecular data showed that the annulata group is considered a member of the genus Platycnemis, as originally proposed. The genus Coeliccia, a member of the subfamily Calicnemiinae (Platycnemididae), is not grouped with the Platycnemidinae. The Disparoneurinae of the ‘Protoneuridae’ showed a closer relationship to the Platycnemidinae than the Calicnemiinae. The dataset supports the placement of the Disparoneurinae as a subfamily of the Platycnemididae. This resolves the monophyly of Platycnemididae.

Advancing the taxonomy of economically important red seaweeds (Rhodophyta)

ABSTRACT The cultivation of red seaweeds for food (nori), agar and carrageenans is the basis of a valuable industry. However, taxonomic knowledge of these cultivated seaweeds and their wild relatives has not kept pace with advances in molecular systematics despite the fundamental importance of being able to identify commercially important species and strains, discover cryptic and endemic taxa and recognize non-native species with potentially damaging diseases and epiphytes. This review focuses on molecular taxonomic advances in the cultivated red algae with the highest commercial value globally: Eucheuma and Kappaphycus, Porphyra sensu lato and Gracilaria. All three groups are similarly taxonomically challenging: speciose, morphologically plastic, with poorly resolved species boundaries. Eucheuma and Kappaphycus are frequently misidentified and the molecular markers cox2-3 spacer, cox1 and RuBisCO spacer have helped in understanding phylogenetic relationships and identifying new species and haplotypes. In Porphyra sensu lato (Bangiales) species identification and phylogenetic relationships were highly problematic until a taxonomic revision based on a two-gene phylogeny (18S and rbcL) resulted in nine genera of bladed species. Pyropia, with at least 89 species, three in nori cultivation, has potential for new commercial evaluation. In Gracilaria sensu lato, earlier efforts to resolve species-level taxonomy and generic descriptions were superseded by application of molecular tools, including DNA sequences of the RuBisCO spacer, rbcL gene, 18S and the ITS region. Studies of these cultivated red algal genera highlight the need for a robust taxonomy, a more standardized approach to the molecular markers used and a comprehensive dataset for each representative species. Current work on DNA-based species delimitation, the emergence of high throughput sequencing, multi-gene phylogenies, publication of whole genomes (e.g. Porphyra umbilicalis) and genomes in the pipeline (e.g. Gracilaria) are increasingly improving our understanding of phylogenomic relationships and species relationships. This knowledge, in turn, can then be applied to improving red seaweed aquaculture.

Biodiversity, Biogeography and Molecular Genetics of the Commercially Important Genera Kappaphycus and Eucheuma

Through the years, several strains and color morphotypes of Kappaphycus and Eucheuma have emerged in cultivation areas as a result of environmental and anthropogenic factors. This led to confusion in nomenclature of these seaweeds. However, the advent of molecular technology has brought notable changes to seaweed research over the past two decades. New molecular techniques have proved useful, particularly in the molecular identification and cataloging of economically important carrageenophytes, i.e. Kappaphycus and Eucheuma which were commercially introduced into many countries worldwide. The following chapter discusses the application and findings of molecular studies within the context of these red seaweeds, along with potential areas for future research.