The effect of temporal variability in salinity on the invasive red alga Gracilaria vermiculophylla

Non-native, invasive species are often characterized by being tolerant to environmental stressors, leaving them more fit relative to native species. The red alga Gracilaria vermiculophylla originates from the NW Pacific but has recently spread along the coastlines of Western Europe, where it has become abundant in many shallow, soft-bottom estuaries. Salinity is important for the local and regional distribution of algae. The distribution of G. vermiculophylla in Europe suggests that it thrives well in hyposaline environments and that it may be more fit than some native algae under such conditions. Little, however, is known about the ecophysiology of G. vermiculophylla and it is therefore difficult to predict its spread and future distribution. Laboratory experiments with G. vermiculophylla showed that steady-state salinity above 15?psu was optimal for growth and that the growth rate was reduced at salinities below 15?psu. Variable salinity reduced the growth rate and larger oscillations were more stressful than small ones. Exposure to very low salinity (0–5?psu) was stressful for the alga and algae exposed to these low levels for 2–4 days were unable to recover fully. Gracilaria vermiculophylla did not seem to perform better in hyposaline conditions than many native, estuarine species. The present distribution of G. vermiculophylla in Scandinavia can be explained well by its response to salinity, but this may not explain its present success relative to many naturally occurring algal species.     

Agar properties of two species of Gracilariaceae from the Gulf of California,Mexico

Agar properties of two potentially commercial important seaweeds from the Gulf of California were studied. Maximum yield in Gracilaria vermiculophylla (45.7%) occurred during the summer months, coinciding with high water temperatures (31°C) whereas minimum yields (11.6%) were obtained during the coldest months of the year when populations of this species diminish in the bay. Gracilariopsis longissima showed two yield peaks, one in spring and another in fall, before the maximum and minimum seawater temperatures. Gel strength in native agar from the two species was low (<22.5 g cm⁻²) for most of the year. G. vermiculophylla native agar showed a slight increase in gel strength from June to August, which were the hottest months. Maximum value was 85 g cm⁻¹ in August. Maximum gel strength in G. longissima was observed in October (91 g cm⁻¹), and an unusual native agar with no detectable gel strength was observed in March and April samples. Gelling temperatures range from 27.7 to 36.5°C in G. vermiculophyla and from 26.6 to 34.9°C in G. longissima, meanwhile melting points were 73.9 – 53.5°C and 75.5 – 56.6°C, respectively. Sulfate content was high, 6.3–13.9% in G. vermiculophylla and 1.9–11.9% in G. longissima, and on the other hand 3,6 anhydrogalactose content was low 12.1–26.7% and 9.1–23%, respectively compared to other species. Results obtained showed that mean native agar yields of Gracilaria vermiculophylla and Gracilariopsis longissima from the Gulf of California are comparable to other tropical Gracilaria. However, the low gel strength, high sulfate content and low 3,6 anhydrogalactose content observed in the native agar extracted from these species make this an agaroid, thus alternative methods of extraction should be used to evaluate the possibility of commercial utilization of both species.     

Gracilaria vermiculophylla: A western Pacific species of Gracilariaceae (Rhodophyta) first recorded from the eastern Pacific

We report Gracilaria vermiculophylla (Ohmi) Papenfuss from the Pacific coast of North America based on a morphoanatomical revision and comparison of sequences of the nuclear gene coding for the small subunit of ribosomal RNA and the internal transcribed spacers of populations from Baja California, Mexico and Hiroshima, Japan. It is the first convincing report of this species out of its center of distribution in western north Pacific, where it has been considered as a synonym of former ‘G. verrucosa’ records. G. vermiculophylla also occurs in central California as indicated by internal transcribed spacer sequences of a previously unknown Gracilaria Greville material. In the northeastern Pacific G. vermiculophylla is characterized by a robust somewhat vermiform, well‐branched cylindrical thallus, with gradual cell size transition from cortex to medulla, deep spermatangial conceptacles, regular chains of carposporangia and carposporangial initials, downwardly oriented tubular nutritive cells, but rarely with upwardly oriented tubular nutritive cells. In some features of its cystocarp anatomy G. vermiculophylla is related to Gracilariopsis E. Y. Dawson or Hydropuntia Montagne and it is a relevant species for discussions about Gracilariaceae genera.     

GRACILARIA VERMICULOPHYLLA (RHODOPHYTA,GRACILARIALES) IN THE VIRGINIA COASTAL BAYS,USA: COX1 ANALYSIS REVEALS HIGH GENETIC RICHNESS OF AN INTRODUCED MACROALGA

Gracilaria vermiculophylla (Ohmi) Papenfuss is an invasive alga that is native to Southeast Asia and has invaded many estuaries in North America and Europe. It is difficult to differentiate G. vermiculophylla from native forms using morphology and therefore molecular techniques are needed. In this study, we used three molecular markers (rbcL, cox2‐cox3 spacer, cox1) to identify G. vermiculophylla at several locations in the western Atlantic. RbcL and cox2‐cox3 spacer markers confirmed the presence of G. vermiculophylla on the east coast of the USA from Massachusetts to South Carolina. We used a 507 base pair region of cox1 mtDNA to (i) verify the widespread distribution of G. vermiculophylla in the Virginia (VA) coastal bays and (ii) determine the intraspecific diversity of these algae. Cox1 haplotype richness in the VA coastal bays was much higher than that previously found in other invaded locations, as well as some native locations. This difference is likely attributed to the more intensive sampling design used in this study, which was able to detect richness created by multiple, diverse introductions. On the basis of our results, we recommend that future studies take differences in sampling design into account when comparing haplotype richness and diversity between native and non‐native studies in the literature.     

GENETIC DATA HINT AT A COMMON DONOR REGION FOR INVASIVE ATLANTIC AND PACIFIC POPULATIONS OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA)1

Gracilaria vermiculophylla (Ohmi) Papenf., an agar‐producing red alga introduced from northeast Asia to Europe and North America, is often highly abundant in invaded areas. To assay its genetic diversity and identify the putative source of invasive populations, we analyzed the mitochondrial cytochrome c oxidase subunit I (cox1) gene from 312 individuals of G. vermiculophylla collected in 37 native and 32 introduced locations. A total of 19 haplotypes were detected: 17 in northeast Asia and three in Europe and eastern and western North America, with only one shared among all regions. The shared haplotype was present in all introduced populations and in ～99% of individuals in the introduced areas. This haplotype was also found at three native locations in east Korea, west Japan, and eastern Russia. Both haplotype and nucleotide diversities were extremely low in Europe and North America compared to northeast Asia. Our study indicates that the East Sea/Sea of Japan is a likely donor region of the invasive populations of G. vermiculophylla in the east and west Atlantic and the east Pacific.     

Taxonomic Considerations of a Foliose Grateloupia Species from the Straits of Messina

Grateloupia turuturu Yamada is the currently accepted name for the invasive red alga that is present on coasts of the North Atlantic. Previously considered as G. doryphora (Montagne) M.A. Howe, populations of this invasive species were examined and their taxonomic position revised using molecular and morphological techniques. It was also thought that similar invasive populations in the Mediterranean should be identified as G. turuturu. This investigation used rbcL based molecular analyses to clarify the taxonomic position of Grateloupia “doryphora’ from the Straits of Messina. Our results indicate that this population is neither G. doryphora nor G. turuturu. It was placed separately in all analyses and grouped consistently with other Grateloupia species from the Pacific. On the basis of molecular data from this and previous investigations, it is evident that the status of the foliose Atlantic and Mediterranean entities is still unclear and a re-evaluation of the old names connected to them should be undertaken.     

Molecular Systematics and Phylogenetics of Gracilariacean Species from the Mediterranean Sea

Mediterranean reports of Gracilariaceae species, in particular those assigned to the G. verrucosa complex, were re-examined with the use of molecular tools, in order to verify their systematic position and better understand their distribution. Within this complex, we recognized four distinct taxa: Gracilariopsis longissima, Gracilaria gracilis, Gracilaria longa and a possible new species. The rbcL gene sequences, together with those of other terete Mediterranean entities, were included in a broad molecular phylogeny of the family. The reproductive characters of the studied taxa do not fit completely with published hypothesis on the generic and intrageneric relationships, suggesting that the anatomy of some subgroups should be better characterized.     

Stress tolerance of the invasive macroalgae <Emphasis Type="Italic">Codium fragile</Emphasis> and <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> in a soft-bottom turbid lagoon

Invasive species are often hypothesized to have superior performance traits. We compared stress tolerance (as change in biomass) of the invasive macroalgae Codium fragile ssp. tomentosoides and Gracilaria vermiculophylla to the native macroalgae Fucus vesiculosus, Agardhiella subulata, Hypnea musciformis and Ulva curvata in Hog Island Bay, a shallow lagoon in Virginia, USA. We hypothesized that the success of the two aliens is due to their high tolerances of turbidity, sedimentation, desiccation, grazing and nutrient enrichment. Like many lagoons, Hog Island Bay is characterized by extensive intertidal mudflats, high turbidity and sedimentation, and high densities of omnivorous mud snails. Nutrient enrichment may also become a problem as land use practices in adjacent watersheds change. Contrary to our hypothesis, C. fragile was less resistant to sedimentation, desiccation and grazing than other algae and had low growth at all light and nutrient levels. This suggests that any superior performance of this invasive species compared to native algae is probably limited to microhabitats where stress is minimal and where bivalve shells facilitate recruitment and long-term persistence. In contrast, G. vermiculophylla was resistant to desiccation, burial and grazing, and was not negatively influenced by either high or low light or nutrient levels. These traits reflect the current success of G. vermiculophylla in already invaded lagoons and estuaries, and indicates that it will likely continue its spread in European and North American turbid and tidal soft-sediment systems.     

Seasonal variation of the agar quality and chemical composition of Gracilaria veleroae and Gracilaria vermiculophylla (Rhodophyceae,Gracilariaceae) from Baja California Sur,Mexico

Yield and physico‐chemical properties of agar from Gracilaria veleroae E.Y. Dawson and Gracilaria vermiculophylla (Ohmi) Papenfuss were studied and the chemical composition of the two seaweeds was determined. Samples were collected seasonally from summer 2003 to spring 2005. The agar yield did not vary significantly between seasons for both species. The lowest agar gel strength was obtained from G. veleroae (207.5 g cm^–2) in summer 2003 and the highest from G. vermiculophylla (793.1 g cm^–2) in winter 2004. Melting temperatures and hysteresis were higher in G. vermiculophylla, whereas gelling temperatures and 3,6‐anhydrogalactose content were higher for G. veleroae. Moisture, ash, crude fiber, and ether extract showed no significant seasonal variation for G. veleroae. The chemical composition of G. vermiculophylla showed significant seasonal variation. G. vermiculophylla possesses a better agar quality than G. veleroae and is a species that could be considered as a source of agar for commercial use.     

Distribution and ecological role of the non-native macroalga Gracilaria vermiculophylla in Virginia salt marshes

Intertidal salt marshes are considered harsh habitats where relatively few stress-resistant species survive. Most studies on non-native species in marshes describe terrestrial angiosperms. We document that a non-native marine macroalga, Gracilaria vermiculophylla, is abundant throughout Virginia’s Atlantic coastline. We sampled eight marshes, characterized by low slopes and by the presence of the tube-building polychaete Diopatra cuprea on adjacent mudflats, which have been shown previously to be associated with G. vermiculophylla. G. vermiculophylla was found in 71% of the sampled quadrats on the border between the mudflat and tall Spartina alterniflora, 51% within the tall S. alterniflora zone, and 12% further inland. We also tagged G. vermiculophylla from two habitats: (1) unattached G. vermiculophylla within marshes and (2) G. vermiculophylla ‘incorporated’ onto D. cuprea tubes on the adjacent mudflats. Of the incorporated thalli, 3–9% ended up in the marsh, demonstrating connectivity between habitats. In addition, 21% of unattached thalli remained for 2 weeks within the marsh, suggesting that entanglement around marsh plants reduces tidal drift. Growth experiments in mesh bags indicate that most of the G. vermiculophylla transferred from the lagoon to the marsh decomposed there, potentially enhancing local nutrient levels. Finally, we document that G. vermiculophylla in marshes had a reduced associated flora and fauna compared to G. vermiculophylla on the adjacent Diopatra mudflats. In conclusion, unattached G. vermiculophylla is abundant along marsh borders in the tall S. alterniflora zone in Virginia, and we hypothesize that this non-native species has significant impacts in terms of marsh habitat complexity, species abundance and diversity, nutrient dynamics, productivity, and trophic interactions.     

Genetic variation in photosynthetic characteristics among invasive and native populations of reed canarygrass (Phalaris arundinacea)

With the extensive spread of invasive species throughout North America and Europe there is an urgent need to better understand the morphological and physiological characteristics of successful invasive plants and the evolutionary mechanisms that allow introduced species to become invasive. Most ecological studies have focused on morphological differences and changes in community dynamics, and physiological studies have typically explored the differences between native and invasive species. In this study, 15 different genotypes of Phalaris arundinacea from both its native (European) and invasive (North American) range were grown in a common garden experiment to monitor the physiological differences between native and invasive genotypes. Here we present data that suggests high variability exists in the physiological traits among genotypes of P. arundinacea, yet genotypes from the native range are not necessarily physiologically inferior to the hybridized invasive genotypes. Previous work has shown that multiple introductions of P. arundinacea from various European locations to the United States resulted in numerous hybridization events, yielding more genetic variability and phenotypic plasticity in the invasive range. Of the genotypes studied, both morphological and physiological traits of genotypes with French origin were significantly different from the plants from the Czech Republic, North Carolina, and Vermont. The lack of clear differences between native and invasive genotypes indicates that physiological traits may be highly conserved in P. arundinacea and enhanced photosynthetic rates are not indicative of successful invasive genotypes. Instead, morphological traits and defensive secondary compound metabolism may play a more important role in the success of P. arundinacea within its invasive range, and patterns of genetic variation in physiological traits between invasive and native range may be more important than the mean traits of each region when explaining reed canarygrass’ invasive potential in North America.     

FIRST RECORD OF GRACILARIA VERMICULOPHYLLA (GRACILARIALES,RHODOPHYTA) IN THE PO DELTA LAGOONS,MEDITERRANEAN SEA (ITALY)1

Molecular outcomes led us to report the first occurrence of the invasive alien species Gracilaria vermiculophylla (Ohmi) Papenf. in the Mediterranean Sea. This species was recorded for the first time in the Po Delta lagoons in May and October 2008, probably introduced by the importation of the Manila clam Tapes philippinarum. At present, G. vermiculophylla is spread only near some clam‐farming areas, but its diffusion is expected to increase with the colonization of other lagoons where aquaculture is spread, as already observed for other alien species such as Agardhiella subulata and Solieria filiformis. The present study supplies further information on the morphology of this species, the ecological characteristics of the colonized areas, and the most probable introduction vector, confirming that the species spreading occurs in eutrophic and turbid coastal systems.     

Comparison of three gracilarioids: growth rate, agar content and quality

Three gracilarioid species, Gracilariopsis bailiniae and Gracilaria tenuistipitata from Vietnam and Gracilaria gracilis from Russia, were studied in order to determine whether Gracilaria gracilis might be a superior species for cultivation in brackish-water ponds for agar production compared with the Vietnamese species. The effects of different salinity levels on the growth rate and agar production as well as agar properties of three gracilarioid species were compared in controlled laboratory experiments. Gracilaria tenuistipitata and G. gracilis were tolerant to low salinity (∼10‰), whereas Gp. bailiniae died under these conditions. G. tenuistipitata showed superior growth among the three species examined. Gracilaria gracilis had the highest agar content [36.8–46.6% dry weight (dw)]. Agar yield from Vietnamese gracilarioids did not exceed 30% dw. Gel strength of native agar from Gracilaria gracilis was two-fold higher that from Vietnamese species (278 g cm⁻² vs 130 g cm⁻²). Alkali pretreatment increased gel strength significantly for Gracilaria gracilis (1.4-fold), and G. tenuistipitata and Gp. bailiniae (2.3-fold) compared with native agar. The results suggest that Gracilaria gracilis may be a suitable species for production of reasonably good quality agar.     

Economically important seaweeds in Mar Piccolo,Taranto (southern Italy): a survey

A preliminary survey was carried out of the marine algae in the Mar Piccolo Basin, Taranto (southern Italy), in order to assess the degree of sewage pollution and to determine the presence of any economically important species that might be productively utilized. The following commercial species, belonging to a drift algal community, were found: Gracilaria bursapastoris, G. cf. verrucosa, Solieria sp. and S. filiformis. The species showed an aggregated distribution within the community and a clear cyclic annual pattern. Gracilaria bursa-pastoris exhibited the highest mean biomass values in May, whereas Solieria spp. were the most abundant in October. At the present time, only the harvesting of G. bursa-pastoris appears profitable. However, further detailed studies are required to more fully assess the standing crop of the Solieria spp. and G. cf. verrucosa. Considering the environmental situation in the basin, it might be more appropriate to cultivate, rather than harvest, these commercially important seaweeds. With the large number of sewage outlet plants available in the basin, the cultivation of seaweeds in a wastewater treatment-aquaculture system should be considered.     

Dispersal Behavior, Boldness, and the Link to Invasiveness: A Comparison of Four Gambusia Species

Dispersal is a key element of a species' invasiveness. Although considerable work has addressed how dispersal influences the pattern of spatial spread of invading organisms, few studies investigate whether invasive species are in fact better dispersers than either the species they displace or less successful invaders. Recent work suggests that variation in dispersal may be due to variation in an underlying behavioral trait, boldness. Our study examined the link between dispersal, boldness, and invasiveness by comparing the dispersal characteristics and refuge use of two invasive Gambusia species to two congeners in experimental streams. The streams consisted of a series of pools (no flow) connected to a flowing channel. For each species, small groups of females were released at the middle pool, and their movement and activity were recorded over a 1-h period. We found invasive Gambusia to be more likely to disperse out of the introductory pool, to disperse sooner, to travel a greater distance in the artificial streams, and thus to exhibit greater dispersal tendencies than their close relatives. Among the invasives, Gambusia affinis had a greater dispersal tendency than G. holbrooki. We suspect this result indicates variation in the contribution of dispersal to the relative invasiveness of these species. Certain dispersal measures were correlated to time spent out of refuge, although invasive Gambusia and their relatives did not differ in the predicted manner. These results argue for the greater incorporation of experimental approaches and analyses of behavioral mechanisms in the study of invasive species.     

North American History of Two Invasive Plant Species: Phytogeographic Distribution, Dispersal Vectors, and Multiple Introductions

The North American historic phytogeographic distribution of mugwort (Artemisia vulgaris) and Japanese knotweed (Polygonum cuspidatum), two invasive perennial species introduced from Eurasia and East Asia respectively, was recreated using herbarium records. The putative initial introduction of these two species differs by c.a. 400 years, but their patterns of geographic distribution, introduction pathways, and local dispersal pathways are similar. Both species showed the expected logistic growth relationship between range size and the time following introduction, with lag phases of nearly 400 and 50 years for mugwort and Japanese knotweed respectively. The intrinsic growth rate was greater in Japanese knotweed than mugwort for the US, Canada, and North America. Both species were frequently found along waterway, railroad, and road rights-of-way. Introduction pathways differed, with Japanese knotweed commonly labeled as an ornamental escape (151 collections), while mugwort was commonly cited as an inadvertent component of ship ballast (20 collections). These potential founding populations were located across the final distribution for both species, suggesting anthropogenic large-scale dispersal across North America with local secondary spread. Range expansion appears to be active for both species in the US while nearing the carrying capacity in Canada. Managers of mugwort and Japanese knotweed can make use of this information on their range expansion dynamics and dispersal pathways by reducing anthropogenic dispersal and focusing resources on satellite populations and invasion corridors.     

Photosynthetic and respiratory responses of Gracilaria vermiculophylla (Ohmi) Papenfuss collected from Kumamoto, Shizuoka and Iwate, Japan

The photosynthetic and respiratory responses to irradiance, salinity and temperature of the red alga, Gracilaria vermiculophylla, collected from Kumamoto, Shizuoka and Iwate in Japan were studied using an electronic Dissolved Oxygen sensor. The parameters derived from the photosynthesis versus irradiance relationship indicated the potential to acclimate to broad irradiance variations in all of the populations of G. vermiculophylla collected from these three sites. In addition, the light-saturated photosynthesis rate (P _max) and the dark respiration rate of all populations increased with increasing temperature up to 20–30°C, while the P _max decreased at 35°C. All populations also showed a broad variation of photosynthetic responses to salinity changes in the range from 10 to 30 psu. On the other hand, the population from Iwate showed high photosynthetic efficiency, especially in the temperature range of 5–10°C, and showed low values of saturation irradiance compared to the populations from Shizuoka and Kumamoto. These results suggest that there is greater potential to acclimate to low irradiance and low temperature in the population from Iwate compared to those from the Shizuoka and Kumamoto populations. However, the P _max of the populations from Iwate and Shizuoka was reached at 20°C and 25°C, respectively, while the Kumamoto population reached P _max at 30°C. This implies that the latter population has greater potential to tolerate higher temperatures than the former. Such characteristics in photosynthesis and respiration of G. vermiculophylla collected from the three locations probably indicate an acclimation to prevailing environmental conditions in their respective habitats.     

Flora and fauna associated with the introduced red alga Gracilaria vermiculophylla

This paper presents the first detailed study of the spread of the introduced marine red alga Gracilaria vermiculophylla on the west coast of Sweden, and of the fauna and flora associated with this alga in Scandinavia and the western mid-Atlantic. G. vermiculophylla was discovered in the archipelago of Göteborg, Sweden, in the summer of 2003, and in 2005 its distribution range covered at least 150 km. The species is typically found as loose-lying thalli or attached to small stones and mollusc shells within low-energy bays and estuaries. Both gametophytic and tetrasporophytic specimens were found, as well as specimens with mixed reproductive stages. In order to assess the importance of this introduced alga as a habitat for native benthic organisms, attached and loose-lying individuals of G. vermiculophylla were sampled from invaded locations in Sweden, Denmark and Virginia (United States). In total we found 92 taxa associated with G. vermiculophylla. The dominant classes were Malacostraca, Gastropoda and Florideophyceae. The diversity of the associated taxa was not affected by attachment status, or G. vermiculophylla biomass. In Virginia and Sweden animal abundances were positively correlated with the biomass of algae and plants associated with G. vermiculophylla. If G. vermiculophylla primarily invades non-vegetated soft-sediment estuaries, the invasion may lead to an increase in abundances of small native invertebrates (e.g. gastropods and crustaceans) and epiphytic algae, with likely cascading effects on higher trophic levels.     

Growth,photosynthesis and agar in wild-type strains of Gracilaria verrucosa and G. conferta (Gracilariales,Rhodophyta), as a strain selection experiment

The local species Gracilaria conferta and the foreign G. verrucosa were grown together under a wide range of photon flux density and temperature conditions. Gracilaria verrucosa showed a higher growth rate, especially under low temperatures, and higher photosynthetic performances as well as higher ribulose-1,5-bisphosphate carboxylase activity as compared with G. conferta. Gracilaria verrucosa also showed a better quality and yield of agar, suggesting that this species could be more suitable than G. conferta for outdoor cultivation in Israel and may improve winter growth in ponds. Growth rate and agar quality (gel strength) were rated as the most suitable characteristics influencing the preference of strains for outdoor cultivation.     

GRACILARIA HUMMII SP. NOV. (GRACILARIALES,RHODOPHYTA), A NEW NAME FOR THE AGAROPHYTE “GRACILARIA CONFERVOIDES” HARVESTED IN NORTH CAROLINA DURING WORLD WAR II1

Gracilaria hummii Hommers. et Freshwater is proposed as a new name for the inshore cylindrical species found in North Carolina that was treated as Gracilaria confervoides (L.) Grev. during World War II, and more recently as G. verrucosa (Huds.) Papenf. Molecular evidence places G. hummii in the Gracilis‐group in Gracilaria together with G. gracilis (Stackh.) Steentoft, L. M. Irvine et Farnham, the name currently applied to specimens formerly identified as G. confervoides and G. verrucosa. G. hummii differs from G. gracilis in possessing shallower male conceptacles in which the spermatangial filament originates from a surface cortical cell rather than from a subcortical cell. The cystocarps are similar, except that the gonimoblasts of G. hummii are attached to the base of the pericarp by numerous, prominent thickened terminal tubular cells and because terminal tubular cells are absent above the midregion of the cystocarp or in the vicinity of the ostiole. The gonimoblasts are subtended by a bundle of longitudinally oriented, thick‐walled secondary filaments of a type that has not been described before in Gracilaria but that may be characteristic of some other species. G. hummii occupies a basal position in the Gracilis‐group and is distinct from all other cylindrical North Carolina Gracilaria species, according to the molecular and morphological evidence.