Reinstatement of Grateloupia subpectinata (Rhodophyta, Halymeniaceae) based on morphology and rbcL sequences

Morphological observations and molecular analyses of the north‐western Pacific species of the red algal genus Grateloupia (Halymeniaceae) indicate the presence of an entity, which is somewhat similar in gross morphology to G. asiatica Kawaguchi et Wang but is distinguished from the latter species by some morphological features. These include: (i) a somewhat fleshy texture; (ii) wider and much thicker (4.5–10 mm wide and up to 1300 μm thick) axes, of which an inner cortex consists of more (6–9) cells; (iii) generally longer (up to 17 cm), marginal and surface proliferations that are clearly constricted (terete) at bases; and (iv) much elongated, oblong auxiliary cells. Phylogenetic analysis using the ribulose‐l,5‐bisphosphate carboxylase/ oxygenase (rbcL) gene of G. asiatica and the alga in question shows them to be distantly related and strongly supports the differentiation of these two entities at the species level. Judging from the literature, this entity is actually Grateloupia subpectinata Holmes, which has been placed into synonymy under G. asiatica [as G. filicina (Lamouroux) C. Agardh] or G. prolongata J. Agardh in previous reports, and therefore the Holmes name is reinstated.     

SYSTEMATICS OF GRATELOUPIA FILICINA (HALYMENIACEAE,RHODOPHYTA), BASED ON rbcL SEQUENCE ANALYSES AND MORPHOLOGICAL EVIDENCE,INCLUDING THE REINSTATEMENT OF G. MINIMA AND THE DESCRIPTION OF G. CAPENSIS SP. NOV.1

Grateloupia filicina (C. Agardh) Lamouroux, originally described from the Mediterranean Sea, has long been considered a textbook example of a marine red alga with a cosmopolitan distribution. An rbcL‐based molecular phylogeny, encompassing samples covering the entire geographic distribution of the species, revealed a plethora of “cryptic” species, whereby the presence of genuine G. filicina is limited to the Mediterranean basin. The phylogeny revealed a strong biogeographic imprint, with specimens from temperate regions resolved in clades composed of species inhabiting the same geographic region. Presence of widely divergent morphologies in the temperate clades indicated that several lineages have converged independently to a G. filicina‐type morphology. Tropical representatives are resolved in a single clade with very uniform G. filicina‐type morphology and pairwise sequence divergences that are lower than the average divergence observed in temperate lineages. This, combined with a lack of clear geographic structure among the tropical lineages, may indicate a more recent divergence with long‐range dispersal capacities. Violations to the biogeographic signal in temperate lineages seemed to be due to either inadequate taxonomy or recent introductions. Grateloupia minima P. & H. Crouan, a taxon placed in synonymy under G. filicina, is reinstated as a separate species distributed in the northeast Atlantic Ocean. Grateloupia capensis sp. nov. is described to accommodate specimens from South Africa with a G. filicina‐type morphology, and G. filicina var. luxurians is elevated to species status. Morphological and anatomical characters were put forward that support the distinctiveness of these three distinct species.     

Grateloupia Serra sp. nov. H. W. Wang & Y. Lou (Halymeniaceae,Rhodophyta): a new species previously confused with Grateloupia asiatica in China

A new Grateloupia species from Luxun park, Qingdao Province, North China, was discovered during recent investigations and named Grateloupia serra H. W. Wang &; Y. Lou sp. nov. Morphological observations showed that: (1) the thalli were purple to dark red, cartilaginous and mucilaginous in texture, 15–45?cm in height; (2) the surface of thalli was covered with numerous proliferous branchlets and proliferous branchlets that were dentate when on the main axes; (3) the thalli were 450–550?µm thick, a cortex consisted of 6–8 layers of oblong or rounded cells and a medulla covered by compact medullary filaments; (4) the carpogonial branch was 6-celled and the auxiliary-cell branch was 5-celled, they were typical Grateloupia-type; (5) the internal structure of mature tetrasporangia were cruciately divided, oblong or square in shape. The morphological differences were supported by molecular analyses based on ribulose-1, 5-bisphosphate carboxylase/oxygenase gene (rbcL) sequences. Sequences of four G. serra sp. nov. samples were embedded into the Grateloupia clade and showed no pairwise divergence.     

INFLUENCE OF POLYAMINES ON THE SPORULATION OF GRATELOUPIA (HALYMENIACEAE,RHODOPHYTA)1

Polyamines (PAs) such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are ubiquitous aliphatic amines involved in widely varying physiological behavior, but particularly they are actively involved in cell growth, division, and differentiation during reproductive events in plants. The contents of PUT, SPD, and SPM in infertile and fertile thalli of the red macroalga Grateloupia sp. were compared, and the results revealed a significant decrease in quantity from infertile to fertile status. At the enzymatic level, l ‐ornithine decarboxylase (ODC) was mainly detected, and l ‐arginine decarboxylase activity was not diminished by the inhibition of ODC. The maximum enzymatic activities, within the range of activities observed, correlated with the lower levels of polyamines in fertile thalli. In culture, SPM promoted the maturation of cystocarps to the eventual liberation of spores from aseptic fertile explants. PAs accumulated in cultivated explants as compared with noncultivated, but exogenous SPM addition further increased the endogenous SPM. The addition of berenyl, cordycepin, cyclohexylamine, dicyclohexylamine, and aurintricarboxylic acid blocked the synthesis in culture at the level of PUT, and partially at SPD and SPM synthesis, but the addition of SPM restored the levels of SPD and SPM as SPM accumulated, and they appeared to interconvert each other. The results obtained suggest that the culture in presence of SPM restored a deficient SPM situation in fertile explants, thus promoting sporulation.     

Taxonomic notes on the Halymeniaceae (Gigartinales,Rhodophyta) from Japan. III. Synonymization of Pachymeniopsis Yamada in Kawabata with Grateloupia C. Agardh*

The taxonomic status of the red algal genus Pachymeniopsis (Halymeniaceae) was evaluated based on a morphological study of the type species Pachymeniopsis lanceolata (Okamura) Yamada in Kawabata (=Aeodes lanceolata Okamura). Features used to separate Pachymeniopsis from allied genera such as Aeodes or Grateloupia were found to be inconsistently expressed within even single populations, and P. lanceolata is considered to be best placed in Grateloupia. The two remaining species, P. yendoi and P. elliptica, are judged to be conspecific, with P. elliptica also best placed in Grateloupia. The genus Pachymeniopsis is, therefore, considered synonymous with Grateloupia. The transfer of P. lanceolata to Grateloupia is proposed as Grateloupia lanceolata (Okamura) Kawaguchi, comb. nov. The reinstatement of Grateloupia elliptica Holmes, which includes Pachymeniopsis yendoi as a synonym, is also proposed.     

Morphological observation,life history and rbcL gene sequence analysis of a new species,Grateloupia variata sp. nov. (Halymeniaceae,Rhodophyta) from Qingdao,China

Grateloupia variata sp. nov. H. W. Wang (Halymeniaceae, Rhodophyta) is newly described from Qingdao, China. The results indicated that: (1) thalli are cartilaginous or gelatinous in texture; (2) thalli are constricted at the base with frequent proliferations; (3) the end portion of branches are irregular, wide, flat or ungulate, split; (4) the cortex is 50–80?μm thick, consisting of four to six layers of cells; the medulla is 10–15?μm long and 1–3?μm wide, consisting of densely and irregularly intertwined filaments; (5) six-celled carpogonial ampullae branched and five-celled auxiliary cell ampullae branched; they are of the typical Grateloupia-type; (6) tetraspores and carpospores grow directly into discoid crusts through initial cell division and are of the immediate discal type; (7) Grateloupia variata sp. nov. shows a typical triphasic life history with homotypic gametophytes, carposporophytes and tetrasporophytes, and a typical homotypic alternation of generations; and (8) the rbcL sequences show no pairwise divergence and the species form a small single monophyletic subclade within the Grateloupia clade. Morphological observations, life history and molecular analysis support G. variata as a new species.     

TWO MORPHOLOGICALLY SIMILAR BIOLOGICAL SPECIES: CHONDRUS PINNULATUS AND C. ARMATUS (GIGARTINACEAE,RHODOPHYTA)1

The nature of the relationship between Chondrus pinnulatus (Harvey) Okamura f. pinnulatus and C. pinnulatus f. armatus (Harvey) Yamada et Mikami (Gigartinaceae, Rhodophyta) was investigated by comparative analysis of their biogeography, phenologies, life histories, gross and vegetative morphology, crossability, and upper thermal tolerance. The forma pinnulatus has a more northerly distribution in Japan and adjacent waters, exhibiting adaptation to the cooler regions, whereas the forma armatus has a more southerly range. The latter may be the result of a higher thermal tolerance. Both formae have a Polysiphonia-type life history and are similar in their internal vegetative morphology. They can, however, be distinguished by gross morphology: forma pinnulatus has wide, flattened axes, compressed to flattened ultimate segments and proliferations, while forma armatus has narrow, compressed to subterete axes and subterete to terete ultimate segments and proliferations. These differences persist in laboratory culture. All intraformae crosses were positive, with carpospores from the cross developing into fertile F₁ tetrasporophytes releasing tetraspores that developed into dioecious F₁ gametophytes, the female gametophytes of which formed normal cystocarps. This suggests that members of populations of each forma freely interbreed. Among interformae crosses, only some offspring derived from geographically distant strains bore normal cystocarps in the F₁ female gametophytes. Other crosses showed that interbreeding between populations of these two formae was blocked by various isolating mechanisms: incompatibility, hybrid inviability, and hybrid sterility. Reproductive isolation between f. pinnulatus and f. armatus is virtually complete in wild populations, because hybrid populations have not been found in the wild. In addition, these two entities can be considered biological species that are also referred to the taxonomic species, C. armatus and C. pinnulatus, because they do not overlap with regard to the morphology of the ultimate segments and proliferations. Subtle (but significant) gross morphological differences, partial interfertility between the two species, and deleterious hybridization in the area in which they occur sympatrically suggest that their evolutionary divergence was relatively recent.     

Cultivation of Grateloupia acuminata (Halymeniaceae,Rhodophyta) by regeneration from cut fragments of basal crusts and upright thalli

Regeneration of Grateloupia acuminata Okamura from fragments of basal crusts and young upright thalli was studied in culture. The carpospores and tetraspores develop into basal crusts and these in turn produce upright thalli. When the crusts and the young upright thalli were cut into fragments, the fragments of basal crusts regenerated into new crusts, whereas those of upright thalli formed adventitious filaments. For cultivation in the sea, the cut fragments of basal crusts were inoculated onto oyster shells and synthetic twines of Nori-net and cultured for about one month in the laboratory. The oyster shells and the twines with regenerated crusts were then transferred into the sea from September to December. Many upright thalli developed and grew well during the cold season of the year (December to March).Author for correspondence     

Research note: First report of the Japanese species Grateloupia lanceolata (Halymeniaceae, Rhodophyta) from California, USA

The Japanese red alga Grateloupia lanceolata (Okamura) Kawaguchi was discovered in southern California at Santa Catalina Island in spring 2003 and April 2008 and in central California at the mouth of the Elkhorn Slough in Moss Landing in May, June and July of 2008. The morphology of thalli from both localities agrees with published figures. Sequences from the rbc L gene and the nuclear marker, internal transcribed spacer-1 from Californian G. lanceolata were identical to those from two specimens of G. lanceolata introduced to the Thau Lagoon, Mediterranean France and a specimen from Japan. It is likely that the import of oysters for mariculture played a role in its introduction into California.     

Production and application of filaments of <Emphasis Type="BoldItalic">Grateloupia turuturu</Emphasis> (Halymeniaceae,Rhodophyta)

Filaments from Grateloupia turuturu were obtained through germination of spores, regeneration from fragments of discoid crusts and erect thalli. The rates of filament formation through the three ways were 5.3 ± 1.2%, 100%, and 62.3 ± 5.6%, respectively. Discoid crusts were the best materials for the production of filaments. The obtained filaments were cloned in stationary and aerated culture. The differentiations of filaments were observed. When attached to the substrata, filaments differentiated into discoid crusts from which erect thalli grew, whereas for filaments in suspension culture, some cells in the filaments differentiated into spherical structures that also formed new erect thalli. Moreover, fragments of filaments (< 100 μ m) were seeded onto nori-nets. The regenerated plantlets grew into adult thalli in field cultivation.     

Red alga Grateloupia imbricata (Halymeniaceae), a species introduced into the Canary Islands

Specimens of Grateloupia from Gran Canaria in the Canary Islands were used to molecularly ascertain which of the species has been used in physiological and bio-technological experiments. The rbc L sequence analysis revealed that four out of five analyzed specimens (i.e. those commonly collected for physiological research) formed a monophyletic clade with G. imbricata from Korea, Japan, and China, and were quite different from any other species of the genus. Another sample, which was associated with cage nets used for fish aquaculture, was grouped with G. lanceolata from Japan, though it appears too early as yet to identify it as such. This is, thus, proof of a new introduction of a marine macroalga, since G. imbricata is an Asian species, native to Japan and Korea, in the Canary Islands. The role of international shipping in the introduction of the species is discussed.     

帚状蜈蚣藻的修订研究——基于形态观察和rbcL序列分析

通过形态观察和分子分析相结合的方法,对青岛（模式标本产地）产的帚状蜈蚣藻（Grateloupia fastigiata Li et Ding）进行了重新鉴定,结果表明:①藻体为深紫红色,高9-26 cm,基部呈圆柱状,1-2回不规则羽状分枝,皮层厚度为85-160 μm,由8-11层细胞构成;助细胞生殖枝丛为典型的Grateloupia型,果胞枝生殖枝丛主枝由6个细胞组成,辅助细胞生殖枝丛主枝由5个细胞组成（6cpb-5auxb型）,与亚洲蜈蚣藻（G.asiatica Kawaguchi et Wang）一致。②基于rbcL基因序列构建的系统树显示:本研究的10个帚状蜈蚣藻样本与青岛产的亚洲蜈蚣藻之间无碱基差异,形成独立的进化支,与大连石槽、石庙子产的亚洲蜈蚣藻的碱基差异均为1 bp（0.08%）,与日本、韩国产的亚洲蜈蚣藻的碱基差异分别为2 bp（0.16%）、3 bp（0.24%）,均属于种内差异,与Grateloupia clade I中蜈蚣藻属内其他种的碱基差异为18（1.47%）-76 bp（6.19%）,均属于种间差异。通过形态观察和rbcL基因序列分析,认为帚状蜈蚣藻与亚洲蜈蚣藻为同一种,将帚状蜈蚣藻作为亚洲蜈蚣藻的异名。     

A morphological and molecular assessment of the genus Prionitis J. Agardh (Halymeniaceae, Rhodophyta)

A critical reassessment of the morphological features of two closely related red algal genera, Grateloupia C. Agardh and Prionitis J. Agardh (Halymeniaceae), shows that members of the two genera share very similar reproductive (including the Grateloupia‐type auxiliary‐cell ampullae) and vegetative characters. Diagnostic features hitherto used for distinguishing these two genera, the texture of blades (lubricous to leathery in Grateloupia vs cartilaginous in Prionitis) and the position of reproductive structures (scattered over the entire blade in Grateloupia vs confined to particular portions of the blade in Prionitis), are continuous across some 75 species of both genera, thus making it difficult to draw a clear‐cut distinction between the two genera. In ribulose‐1,5‐bisphosphate carboxylase/oxygenase gene (rbcL) sequence analyses, the species of Grateloupia and Prionitis, including the two generitypes, constitute a large monophyletic clade in the Halymeniaceae. It is therefore proposed that Prionitis be included in the synonymy under Grateloupia and the appropriate combinations are proposed.     

Morphogenetic effects of daylength in Schottera nicaeensis

The S. nicaeensis thallus exhibits a typical secondary heterotrichy: terete perennant creeping axes and flattened annual erect fronds. The prostrate system produces new uprights in autumn, while in late spring the old blades produce marginal terete proliferations, which usually attach themselves to the substratum becoming new creeping axes. The present study demonstrates that the changes in morphogenetic trends are controlled by daylength, regardless of temperature. Both the upright formation and the flattened growth are short day responses, whereas both the terete proliferation outgrowth and the rhizoid production are long day responses. This seems to be the first report on two vegetative photoperiodic responses to different daylengths within a single algal species.     

TWO TYPES OF AUXILIARY CELL AMPULLAE IN GRATELOUPIA (HALYMENIACEAE,RHODOPHYTA), INCLUDING G. TAIWANENSIS SP. NOV. AND G. ORIENTALIS SP. NOV. FROM TAIWAN BASED ON rbcL GENE SEQUENCE ANALYSIS AND CYSTOCARP DEVELOPMENT1

Few species in the genus Grateloupia have been investigated in detail with respect to the development of the auxiliary cell ampullae before or after diploidization. In this study, we document the vegetative and reproductive structures of two new species of Grateloupia, G. taiwanensis S.‐M. Lin et H.‐Y. Liang sp. nov. and G. orientalis S.‐M. Lin et H.‐Y. Liang sp. nov., plus a third species, G. ramosissima Okamura, from Taiwan. Two distinct patterns are reported for the development of the auxiliary cell ampullae: (1) ampullae consisting of three orders of unbranched filaments that branch after diploidization of the auxiliary cell and form a pericarp together with the surrounding secondary medullary filaments (G. taiwanensis type), and (2) ampullae composed of only two orders of unbranched filaments in which only a few cells are incorporated into a basal fusion cell after diploization of the auxiliary cell and the pericarp consists almost entirely of secondary medullary filaments (G. orientalis type). G. orientalis is positioned in a large clade based on rbcL gene sequence analysis that includes the type species of Grateloupia C. Agardh 1822 , G. filicina. G. taiwanensis clusters with a clade that includes the generitype of Phyllymenia J. Agardh 1848 , Ph. belangeri from South Africa; that of Prionitis J. Agardh 1851 , Pr. lanceolata from Pacific North America; and that of Pachymeniopsis Y. Yamada ex Kawab. 1954, Pa. lanceolata from Japan. A reexamination of the type species of the genera Grateloupia, Phyllymenia, Prionitis, and Pachymeniopsis is required to clarify the generic and interspecific relationships among the species presently placed in Grateloupia.     

Transfer of Pterosiphonia pumila Yendo to the genus Symphyocladia (Rhodomelaceae, Rhodophyta)

The red alga Pterosiphonia pumila Yendo (Rhodomelaceae, Ceramiales) has flattened thalli in which there is complete lateral fusion between branches and their parental axes during early stages of development, the laterals only becoming distally free in reproduc-tively mature plants. This results in a leaf-like organization distinct from that displayed by all other species of the genus Pterosiphonia, the members of which have terete to compressed axes that are congenitally fused along only a few (<4.5) proximal segments of the laterals. The organization of P. pumila is, thus, similar to that of species of the genus Symphyocladia, to which it is here transferred as Symphyoctadia pumila (Yendo) Uwai et Masuda, comb. nov. Symphyoctadia pumila is restricted to Japan and Korea and is distinguished from the other three described species of Symphyocladia by its small (<3 cm long) ecorticate thalli and complete lack of vegetative trichoblasts. Symphyoctadia pennata Okamura has a similar suite of characteristics, has been successfully cross-bred with S. pumila in laboratory culture and is, thus, placed in synonymy with S. pumila.     

Phylogenetic relationships and biogeography of Ptilophora (Gelidiales,Rhodophyta) with descriptions of P. aureolusa,P. malagasya,and P. spongiophila from Madagascar

The genus Ptilophora currently includes 16 species occurring mostly in subtidal habitats of the Indo‐Pacific Ocean, but its global diversity and biogeography are poorly understood. We analyzed mitochondrial cox1, plastid rbcL and plastid psbA sequences from specimens collected in southern Madagascar during the 2010 Atimo Vatae expedition and studied their morphologies. Both morphological and molecular data sets demonstrated the presence of five species in southern Madagascar: Ptilophora hildebrandtii, P. pterocladioides, and three new species described here, P. aureolusa, P. malagasya, and P. spongiophila. Ptilophora aureolusa is distinguished by its compound pinnae with uniformly spaced pinnules. Ptilophora malagasya has an indistinct midrib and irregularly spaced pinnules. Ptilophora spongiophila, heavily coated with sponges, has cylindrical to flattened main axes, lateral and surface proliferations, and spatulate tetrasporangial sori. The species of Ptilophora found in Madagascar are endemic, except P. hildebrandtii, which also occurs in eastern Africa. Ptilophora comprises four phylogenetic groups that map to eastern Australia, Japan, western Australia/Southeast Asia/Madagascar/eastern Africa, and Madagascar/eastern Africa/Aegean Sea. Biogeographical analysis revealed that the ancestor of Ptilophora originated in Australia, but most of the species radiated from Madagascar.     

Grateloupia turuturu (Halymeniaceae,Rhodophyta) is the correct name of the non-native species in the Atlantic known as Grateloupia doryphora

Grateloupia doryphora (Montagne) Howe, originally described from Peru, has repeatedly been reported as an invasive species in Atlantic and Mediterranean waters. Various attempts to explain this species' route of introduction have been unsatisfactory. New evidence from comparative rbcL sequence analysis and morphology suggests that this adventive species in the NE and NW Atlantic corresponds with G. turuturu Yamada, originally described from Japan. This provenance follows a well-recognized trend of invasive marine organisms that have colonized the Atlantic Ocean and Mediterranean Sea from Pacific NE Asia.     

对枝蜈蚣藻的修订研究——基于形态特征和基因序列分析

通过形态观察结合rbcL和COⅠ基因序列分析的方法, 对采自广东省汕头市、浙江省温州市和辽宁省大连市的对枝蜈蚣藻Grateloupia didymecladia Li et Ding的11个标本进行了重新鉴定。结果表明: (1)藻体单生或丛生, 红褐色或深红色, 质地黏滑、肉质、衰老时软骨质, 成熟藻体直立, 高15—50 cm, 主枝明显, 扁平, 宽3—15 mm, 厚约1 mm, 末端渐尖, 1—3回羽状分枝。小枝对生或互生, 生长在主枝上或主枝边缘, 基部缢缩, 长短不一, 最长可达15 cm; 配子体为雌雄异体, 果胞枝生殖枝丛和辅助细胞生殖枝丛均为Grateloupia型, 果胞枝生殖枝丛主枝由5个细胞组成, 辅助细胞生殖枝丛主枝由4个细胞组成(5cpb-4auxb型); 四分孢子囊由四分孢子体的内皮层细胞产生, 呈十字形分裂。以上特征均与亚栉状蜈蚣藻G. subpectinata Holmes一致。(2)基于rbcL基因序列构建的系统树显示, 11个样本之间无碱基差异, 形成独立的进化支, 与产于韩国和日本的亚栉状蜈蚣藻G. subpectinata Holmes碱基差异分别为1 bp (0.08%)和2 bp (0.17%), 属于种内差异; 基于COⅠ基因序列构建的系统树显示, 11个样本之间无碱基差异, 形成独立的进化支, 与韩国产的亚栉状蜈蚣藻碱基差异为1 bp (0.18%), 属于种内差异。根据形态、结构及基因序列分析, 确定对枝蜈蚣藻与亚栉状蜈蚣藻为同一种, 根据优先法则, 将对枝蜈蚣藻G. didymecladia Li et Ding作为亚栉状蜈蚣藻G. subpectinata Holmes的同物异名。亚栉状蜈蚣藻为中国新记录种。     

GENETIC VARIABILITY AND POTENTIAL SOURCES OF GRATELOUPIA DORYPHORA (HALYMENIACEAE,RHODOPHYTA), AN INVASIVE SPECIES IN RHODE ISLAND WATERS (USA)1

Grateloupia doryphora (Montagne) M.Howe is an invasive foliose alga that was reported for the first time in Rhode Island, USA in 1997. The population has since increased in size and expanded in range. In this study, the genetic variation and potential sources of the Rhode Island G. doryphora population were examined using three types of molecular markers: randomly amplified polymorphic DNA (RAPD), nuclear internal transcribed spacer (ITS) sequences, and mitochondrial cox2–cox3 intergenic spacer (COX) sequences. No variation was detected in ITS or COX sequences among Rhode Island G. doryphora individuals. RAPDs, however, did reveal genetic variation, although banding patterns were similar, with RAPD genetic distances between individuals ranging from 0.00 to 0.17. The low level of genetic diversity observed within the Rhode Island population may be due to a small founder population or a founder population derived from a genetically uniform source. To identify possible sources of the Rhode Island invasion, individuals from nine geographically diverse populations of foliose Grateloupia were compared. Phylogenetic trees inferred from RAPD distances and ITS and COX sequences had similar topologies; thus there was phylogenetic congruence among these independent loci. The Rhode Island G. doryphora specimens were genetically similar to specimens from G. doryphora populations located in Portsmouth, England; Tholen Island, The Netherlands; and Brittany and Hérault, France. Interestingly, the G. doryphora population in each of these locations is itself due to an introduction event within the past 40 years.