Phylogeography of Simulium siamense Takaoka and Suzuki complex (Diptera: Simuliidae) in Thailand

The phylogeography of Simulium siamense complex was inferred from mitochondrial DNA sequences. A 586‐bp fragment of the cytochrome oxidase I was sequenced for 92 individuals from 13 populations throughout Thailand, representing five cytoforms (A, B, C, F and G). The cytoforms are not genetically different at the molecular level except for cytoform B, which is genetically distinct from the others. This might indicate that cytoform B is a distinct species. Further morphological and molecular work using other genes is needed to clarify this. Our results also argue for the need of integrated approach, both cytological and molecular studies to understanding biodiversity of black flies. The star‐like shape of the mtDNA genealogy is consistent with the sudden population expansion of the mismatch distribution analysis and large negative values of Fu's F_s and Tajima's D‐tests, indicating a population demographic expansion. The expansion time is estimated to be in the late Pleistocene (about 120 000 years ago). Therefore, the overall low level of genetic structure could be due to sharing a recent history. The ancestral haplotype was found in the mountainous area in northeastern Thailand, suggesting that this area could have been the refugium of the species complex during the Pleistocene glaciations. Our results are consistent with previous findings about population expansion in response to the Pleistocene climatic change, thus revealing the importance of this historical event in shaping the genetic structure and diversity of Southeast Asian mainland species.     

Survey of the terrestrial and freshwater molluscan fauna of the Pilliga forest area in northern inland New South Wales,Australia

The Pilliga forest in northern inland New South Wales, Australia, is one of the largest surviving remnants of native forest on the western slopes of the Great Dividing Range. The Pilliga landscape is a challenging environment for molluscs, dominated by dry sclerophyll forest and with limited and largely ephemeral aquatic habitats. A field survey of the area in 2006–2012 identified a surprisingly rich and relatively intact aquatic native molluscan fauna with five species of bivalves in three families and nine species of freshwater gastropods (four families), including some rare species and range extensions. The native land snail fauna comprised 18 species (six families), including an unusually rich pupillid fauna with nine species. Some range extensions are recorded and some species are narrow-range endemics. The distributions of many aquatic and terrestrial species were correlated with geology or soil type. Introduced molluscs were predominantly found in anthropogenic habitats and include two freshwater gastropods (two families) and nine terrestrial snails and slugs (eight families). This study provides insight into the original molluscan fauna of the western slopes prior to landscape-scale agricultural development and provides a benchmark for future reference.     

Phylogeography of the tidewater goby, Eucyclogobius newberryi (Teleostei, Gobiidae), in coastal California

The tidewater goby, Eucyclogobius newberryi, inhabits discrete, seasonally closed estuaries and lagoons along approximately 1500 km of California coastline. This species is euryhaline but has no explicit marine stage, yet population extirpation and recolonization data suggest tidewater gobies disperse intermittently via the sea. Analyses of mitochondrial control region and cytochrome b sequences demonstrate a deep evolutionary bifurcation in the vicinity of Los Angeles that separates southern California populations from all more northerly populations. Shallower phylogeographic breaks, in the vicinities of Seacliff, Point Buchon, Big Sur, and Point Arena segregate the northerly populations into five groups in three geographic clusters: the Point Conception and Ventura groups between Los Angeles and Point Buchon, a lone Estero Bay group from central California, and San Francisco and Cape Mendocino groups from northern California. The phylogenetic relationships between and patterns of molecular diversity within the six groups are consistent with repeated, and sometimes rapid, northward and southward range expansions out of central California caused by Quaternary climate change. Plio-Pleistocene tectonism, Quaternary coastal geography and hydrography, and historical human activities probably also influenced the modern geographic and genetic structure of E. newberryi. The phylogeography of E. newberryi is concordant with phylogeographic patterns in several other coastal California taxa, suggesting common extrinsic factors have had similar effects on different species. However, there is no evidence of a phylogeographic break coincident with a biogeographic boundary at Point Conception.     

Phylogeography and postglacial dispersion of the chub (Leuciscus cephalus) in Europe

A phylogeographic analysis of mitochondrial DNA variation was performed in order to test the hypothesis of a postglacial recolonization of mid- and north-European rivers from a Danubian refuge. Over 345 chub specimens from European rivers covering most of the species' native range were investigated using 600 bp of the cytochrome b gene. Chub in European rivers belong to four highly divergent mitochondrial groups (lineages) differing by mean divergence estimates from 5.2% to 7.89%. These four lineages have a largely allopatric distribution, implying four geographical sets: two Mediterranean, and two north-European sets. This pattern provided strong evidence for: (i) the eradication of this species from most of Europe during maximum ice extent; (ii) its survival in four refugia (Adriatic side of the Balkans, eastern Greece (Aegean Rivers), southern tributaries of the Danube, and periphery of Black and Caspian Seas); (iii) a differential postglacial recolonization of mid- and northern Europe from the last two refugia only; (iv) the occurrence of this recolonization in two steps for the Danubian (western) lineage that entered western Europe (Rhine-Rhone-Loire drainages) during the Riss-Würm interglacial period and survived the last glaciation there before colonizing Garonne, UK and German drainages up to the Elbe during the Holocene; and (v) the occurrence of this recolonization in a single step for the Ponto-Caspian (eastern) lineage that entered the Baltic area as far as the Oder in the Holocene. Both lineages came into contact in the River Elbe without evident mixing.     

Terrestrial activity,movements and spatial ecology of an Australian freshwater turtle,Chelodina longicollis,in a temporally dynamic wetland system

Animal movements, use of space, activity patterns and habitat use are in part determined by proximal factors such as the landscapes they occupy, seasonal or environmental cues and individual attributes such as sex and body size. Using radio‐telemetry and a drift fence, we examined the contribution of these factors to variation in movements, use of space and terrestrial activity in a freshwater turtle, Chelodina longicollis (Testudines: Chelidae), in south‐east Australia. Movements and use of space depended strongly on landscape attributes, while sex and body size were less important. Movements and use of space also varied seasonally and were partly correlated with rainfall. The high overall vagility of turtles, irrespective of sex and adult body size (13.8 ± 2.8 ha (±SE) home range, 2608 ± 305 m total distance moved, 757 ± 76 m range length), probably reflects a common need to be mobile in a landscape characterized by fluctuating resources in temporary wetlands. Use of temporary wetlands also drives C. longicollis into terrestrial habitats for movements between wetlands and extended refuge. Timing of inter‐wetland movements was associated with rainfall, but most notably for immature individuals and for those moving towards temporary wetlands. Movements of adults, evacuation of the drying wetland and periods of extended refuge (i.e. aestivation) were less dependent upon rainfall if at all. We conclude that movements about and use of the landscape by C. longicollis are under the strong influence of several interacting factors such as patch configuration, seasonal and environmental cues, and in part, body size. We argue that such behaviours are also ultimately under selection from the costs and benefits of these behaviours in the context of fluctuating resources.     

Effects of flooding on recruitment and dispersal of the Southern Pygmy Perch (Nannoperca australis) at a Murray River floodplain wetland

Summary With limited evidence linking Australia's Murray‐Darling Basin fish species and flooding, this study assessed annual variation in abundance and recruitment levels of a small‐bodied, threatened floodplain species, the Southern Pygmy Perch (Nannoperca australis), in floodplain habitats (creeks, lakes and wetlands) in the Barmah‐Millewa Forest, Murray River, Australia. Spring and summer sampling over a 5‐year period encompassed large hydrological variation, including 1 year of extended floodplain inundation which was largely driven by an environmental water release, and 2 years of severe regional drought. Recruitment and dispersal of Southern Pygmy Perch significantly increased during the floodplain inundation event compared with the other examined years. This study provides valuable support for an environmental water allocation benefiting a native species, and explores the link between flooding and its advantages to native fish. This suggests that the reduced flooding frequency and magnitude as a result of river regulation may well be a major contributing factor in the species’ decline in the Murray‐Darling Basin.     

Phylogeography of a widespread small carnivore,the western spotted skunk (Spilogale gracilis) reveals temporally variable signatures of isolation across western North America

We analyzed phylogeographic patterns in the western spotted skunk, Spilogale gracilis Merriam, 1890 (Carnivora: Mephitidae) in relation to historical events associated with Pre‐Pleistocene Divergence (PPD) and Quaternary climate change (QCC) using mitochondrial DNA from 97 individuals distributed across Western North America. Divergence times were generated using BEAST to estimate when isolation in putative refugia occurred. Patterns and timing of demographic expansion was performed using Bayesian skyline plot. Putative climatic refugia resulting from Quaternary climate change were identified using paleoecological niche modeling and divergence dates compared to major vicariant events associated with Pre‐Pleistocene conditions. We recovered three major mitochondrial clades corresponding to western North America (California, Baja, and across the Great Basin), east‐central North America (Texas, central Mexico, New Mexico), and southwestern Arizona/northwestern Mexico. Time to most recent common ancestor for S. gracilis occurred ~1.36 Ma. Divergence times for each major clade occurred between 0.25 and 0.12 Ma, with signature of population expansion occurring 0.15 and 0.10 Ma. Ecological niche models identified three potential climatic refugia during the Last Interglacial, (1) west coast of California and Oregon, (2) northwestern Mexico, and (3) southern Texas/northeastern Mexico as well as two refugia during the Last Glacial Maximum, (1) western USA and (2) southern Texas/northeastern Mexico. This study supports PPD in shaping species‐level diversity compared to QCC‐driven changes at the intraspecific level for Spilogale, similar to the patterns reported for other small mammals (e.g., rodents and bats). Phylogeographic patterns also appear to have been shaped by both habitat and river vicariance, especially across the desert southwest. Further, continuing climate change during the Holocene coupled with anthropogenic modifications during the Anthropocene appears to be removing both of these barriers to current dispersal of western spotted skunks.     

Phylogeography of the bigeye chub Hybopsis amblops (Teleostei: Cypriniformes): early Pleistocene diversification and post‐glacial range expansion

The bigeye chub, Hybopsis amblops, is a member of the Central Highlands ichthyofauna of eastern North America. Phylogenetic analyses of the H. amblops species group based on a 1059 bp fragment of the mitochondrial DNA cytochrome b gene did not recover a monophyletic group. The inclusion of Hybopsis hypsinotus in the species complex is questionable. Within H. amblops, five strongly supported clades were identified; two clades containing haplotypes from the Ozark Highlands and three clades containing haplotypes from the Eastern Highlands and previously glaciated regions of the Ohio and Wabash River drainages. Estimates of the timing of divergence indicated that prior to the onset of glaciation, vicariant events separated populations east and west of the Mississippi River. East of the Mississippi River glacial cycles associated with the blocking and rerouting of the Teays River system caused populations to be pushed southward into refugia of the upper Ohio River. Following the most recent Wisconsinan glaciation, populations expanded northward into previously glaciated regions and southward into the Cumberland River drainage. In the Ozarks, west of the Mississippi River, isolation of clades appears to be maintained by the lack of stream capture events between the upper Arkansas and the White River systems and a barrier formed by the Arkansas River.     

Reproductive phenology of river and lake populations of freshwater mussels (Unionida: Hyriidae) in the River Murray

Gonadal cycles are described for Alathyria jacksoni and Velesunio ambiguus from the River Murray at Overland Corner (near Lock 3) and for V. ambiguus at Point Sturt, Lake Alexandrina. The study included periods of low flow (1982), minor flooding (1983) and intermediate flow (1986). In males, gametogenesis continued year-round with fertilisation in late winter. Female A. jacksoni brooded embryos in spring and released glochidia during spring and summer, whereas in female V. ambiguus, glochidia were present most of the year, with peak releases in spring and summer. In both species, the proportions of actively reproducing individuals varied. In the Murray, populations were influenced by variations in discharge. At Point Sturt, gonadal cycles in V. ambiguus were irregular, influenced by variable lake levels. Although both species are essentially gonochoristic, there were hermaphrodites in river and lake populations, and a female bias in the population at Point Sturt. Infections by parasitic trematodes at Point Sturt were rare, but occurred in 10% of A. jacksoni and 35% of V. ambiguus at Overland Corner. The river populations were also affected by gill damage due to unionicolid mites. The disparities in infections between river and lake mussels are unexplained, but did affect their reproductive activity.     

Phylogeography of the cyprinid Squalius aradensis and implications for conservation of the endemic freshwater fauna of southern Portugal

The Iberian cyprinid fauna, characterized by the presence of numerous endemic species, has suffered from significant habitat degradation. The critically endangered Squalius aradensis is restricted to small drainages of southern Portugal, habitats that typically exhibit a characteristic Mediterranean-type heterogeneous hydrological system throughout the year, including alternation of flooding events during winter and complete drought in large river sections during summer. To assess the effect of historical and recent processes on genetic diversity in S. aradensis we examined within- and among-population variability in cytochrome b and six polymorphic microsatellite loci. Estimates of genetic diversity in time and space through the combined use of traditional Phi-/F-statistics, phylogenetic trees, ordination methods and nested clade analysis indicated significant and congruent structuring among populations. Data suggest that the Arade drainage represent the evolutionary centre of the species, with subsequent allopatric fragmentation across drainages. Factors other than isolation by distance strongly affected the within-drainage genetic differentiation observed in these Mediterranean-type drainages, including recent population expansion from a bottleneck event and restricted gene flow imposed by a long-term barrier (brackish water area). Significant correlation was found between S. aradensis allelic diversity and upstream drainage area. The relevance of findings for conservation issues is discussed in relation to local intermittent hydrological conditions, the highly restricted distribution and the critically endangered status of the species.     

Islands of water in a sea of dry land: hydrological regime predicts genetic diversity and dispersal in a widespread fish from Australia's arid zone, the golden perch (Macquaria ambigua)

Rivers provide an excellent system to study interactions between patterns of biodiversity structure and ecological processes. In these environments, gene flow is restricted by the spatial hierarchy and temporal variation of connectivity within the drainage network. In the Australian arid zone, this variability is high and rivers often exist as isolated waterholes connected during unpredictable floods. These conditions cause boom/bust cycles in the population dynamics of taxa, but their influence on spatial genetic diversity is largely unknown. We used a landscape genetics approach to assess the effect of hydrological variability on gene flow, spatial population structure and genetic diversity in an Australian freshwater fish, Macquaria ambigua. Our analysis is based on microsatellite data of 590 samples from 26 locations across the species range. Despite temporal isolation of populations, the species showed surprisingly high rates of dispersal, with population genetic structure only evident among major drainage basins. Within drainages, hydrological variability was a strong predictor of genetic diversity, being positively correlated with spring-time flow volume. We propose that increases in flow volume during spring stimulate recruitment booms and dispersal, boosting population size and genetic diversity. Although it is uncertain how the hydrological regime in arid Australia may change under future climate scenarios, management strategies for arid-zone fishes should mitigate barriers to dispersal and alterations to the natural flow regime to maintain connectivity and the species' evolutionary potential. This study contributes to our understanding of the influence of spatial and temporal heterogeneity on population and landscape processes.     

Phylogeography and diversity of the terrestrial isopod Spherillo grossus (Oniscidea: Armadillidae) on the Australian East Coast

Recent work on terrestrial isopods has shown that morphospecies can have a high degree of genetic diversity. We conducted a molecular phylogenetic study of Spherillo grossus (Budde‐Lund, 1885), a terrestrial isopod endemic to the east coast of Australia. We sequenced the mitochondrial 16S rDNA gene of 63 specimens from 12 collection localities. From a subset of these specimens, we also sequenced the mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 18S rDNA genes. Deep mitochondrial divergences were found among groups of individuals (with p‐distances up to 15, 14, and 0.7% for 16S, COI, and 18S respectively), a pattern consistent with the hypothesis that gene flow between populations has been influenced by the fragmentation of Australia's mesic environment since the Miocene. We also found evidence of human‐mediated dispersal of S. grossus. Scanning electron and light microscopy of a subset of samples provide preliminary evidence that S. grossus is morphologically homogeneous. Our results mirror those found in studies of oniscid isopods from the Northern Hemisphere. © 2013 The Linnean Society of London     

Phylogeography and population structure of the Atlantic and Mediterranean green turtle Chelonia mydas: a mitochondrial DNA control region sequence assessment

Mitochondrial (mt) DNA sequences were analysed to resolve the phylogeography and population genetic structure of Atlantic and Mediterranean populations of green turtles ( Chelonia mydas ). Analysis of sequence variation over 487 base pairs of the control (D-loop) region identified 18 haplotypes among 147 individuals from nine nesting populations. Pairwise comparisons of haplotype frequencies distinguished most nesting colonies, indicating significant genetic differentiation among rookeries and a strong propensity for natal homing behaviour by nesting females. Comparison of control region sequence data to earlier restriction fragment length polymorphism (RFLP) data for the same individuals demonstrates approximately a sixfold higher substitution rate in the 5' end of the control region. The sequence data provide higher resolution both in terms of the number of mtDNA genotype variants and the phylogeographic relationships detected within the Atlantic region, and reveal a gene genealogy that distinguishes two groups of haplotypes corresponding to (i) the western Caribbean and Mediterranean, and (ii) eastern Caribbean, South Atlantic and West Africa. The data suggest that phylogeographic patterns in the Atlantic Ocean may be interpreted in terms of female nest site fidelity and episodic dispersal events. The distribution of mtDNA haplotypes within the region is thus explained by the geological and climatic alternations (glacial and interglacial) over the last million years.     

Distinguishing past from present gene flow along and across a river: the case of the carnivorous marsupial (<Emphasis Type="Italic">Antechinus flavipes</Emphasis>) on southern Australian floodplains

Humans have altered many floodplain ecosystems around the world by clearing vegetation, building towns and regulating river flows. Studies discerning gene flow and population structure of floodplain-dwelling animals are rare yet are necessary for understanding the effects of human actions on native populations. In south-eastern Australia, the yellow-footed antechinus (Antechinus flavipes) is the only carnivorous marsupial on many lowland floodplains, yet our knowledge of impacts of human activities is limited. The control region of mitochondrial DNA and 11 microsatellite DNA markers were used to explore historic and current gene flow in A. flavipes across and along the Murray River. Simulations were carried out to test different migration models. We found evidence for historic gene flow along and across the river but inferred that small towns and farmland or cleared floodplain sections restricted current gene flow along the river. Populations along the river appear to be isolated, and should be maintained at large enough sizes to avoid genetic problems such as inbreeding depression and loss of evolutionary potential. We also investigated whether 50-year-long maintenance of high water levels for irrigation in summer, at the time of juvenile dispersal, has led to restrictions in gene flow across the river. We found no evidence for restrictions to gene flow across the river and suggest that large floods and dropping tree branches may aid dispersal across the river.     

Molecular phylogenetics and morphological variation reveal recent speciation in freshwater mussels of the genera Arcidens and Arkansia (Bivalvia: Unionidae)

Intra‐ and interspecific morphological variation due to both phenotypic plasticity and evolutionary convergence hinder the work of taxonomists and lead to over‐ and underestimates of species richness. Nevertheless, most species on Earth are recognized solely based on morphological characters. We used molecular phylogenetic and morphometric techniques to examine two freshwater mussel species. One is common and widespread, while the other is imperiled and endemic to the Interior Highlands of the USA. Phylogenetic and molecular clock analyses revealed that divergence of Arcidens confragosus and Arkansia wheeleri is small and relatively recent. Divergence in these and other taxa is probably due to isolation of streams in the Interior Highlands. Morphometric analyses showed distinct shell shapes using traditional morphometrics, but not through geometric morphometrics. Outlined shell shapes are indistinguishable; geometric morphometrics could not capture a three‐dimensional component. Our analyses support the validity of these two species as congeners, with the nomen Arcidens (Simpson 1900) having priority. Because shell morphologies are both heritable and environmentally determined, our study emphasizes the importance of considering both molecular and morphometric analyses for identification of freshwater molluscs of conservation concern. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 535–545.     

Length‐weight relationships of three freshwater fish species from the Nandu River and Changhua River in Hainan Island,China

Length‐weight relationships (LRWs) were determined for three fish species from the Hainan Island, China. Fish specimens were sampled across the Nandu River and Changhua River from May 2017 to June 2018 with benthic fyke‐nets (stretched mesh size: 0.4 cm), multi‐mesh gillnets (stretched mesh size ranging from 1.0 to 12.5 cm) and electrofishing. Relationships between length and weight were fitted very well for all species (all R²>0.95). The b values of the LRW equations ranging from 2.938 for Liniparhomaloptera qiongzhongensis to 3.259 for Toxabramis houdemeri.     

Phylogeography and historical demography of the Italian treefrog, Hyla intermedia, reveals multiple refugia, population expansions and secondary contacts within peninsular Italy

We investigated the geographical patterns of genetic diversity in the Italian treefrog through sequence analysis of a mitochondrial cytochrome b gene fragment. Three main mitochondrial lineages were identified, distributed in northern, central and southern Italy, respectively. Their divergence appears indicative of a split time largely predating Late Pleistocene climatic oscillations, and syntopy between them was only observed in the geographically intermediate populations. The historical demographic reconstructions suggest that in both northern and central Italy, an expansion occurred during the last major glacial phase, when a vast widening of the lowland habitats followed the glaciation-induced fall of the sea level. Instead, in southern Italy an expansion event likely followed the end of the last glaciation, although the inference of expansion appears less reliable for the southern clade than for the others. Within this geographical area, a sharp phylogeographic discontinuity separated peninsular from Sicilian populations, and the overall pattern of diversity suggests that the latter derived from a recent colonization of the island, probably through a Late Pleistocene land bridge. Phylogenetic, phylogeographic and historical demographic analyses thus concur in delineating a scenario of multiple refugia, with four groups of populations which survived the last glacial-interglacial cycles in at least three distinct refugia arranged along peninsular Italy, and have recently come into contact following range expansions. Therefore, these results support the hypothesis that a plethora of microevolutionary processes, rather than the prolonged stability of populations, were mainly responsible for shaping the patterns of diversity within this major biodiversity hotspot.     

On the roles of landscape heterogeneity and environmental variation in determining population genomic structure in a dendritic system

Dispersal and natural selection are key evolutionary processes shaping the distribution of phenotypic and genetic diversity. For species inhabiting complex spatial environments however, it is unclear how the balance between gene flow and selection may be influenced by landscape heterogeneity and environmental variation. Here, we evaluated the effects of dendritic landscape structure and the selective forces of hydroclimatic variation on population genomic parameters for the Murray River rainbowfish, Melanotaenia fluviatilis across the Murray–Darling Basin, Australia. We genotyped 249 rainbowfish at 17,503 high‐quality SNP loci and integrated these with models of network connectivity and high‐resolution environmental data within a riverscape genomics framework. We tested competing models of gene flow before using multivariate genotype–environment association (GEA) analysis to test for signals of adaptive divergence associated with hydroclimatic variation. Patterns of neutral genetic variation were consistent with expectations based on the stream hierarchy model and M. fluviatilis’ moderate dispersal ability. Models incorporating dendritic network structure suggested that landscape heterogeneity is a more important factor determining connectivity and gene flow than waterway distance. Extending these results, we also introduce a novel approach to controlling for the unique effects of dendritic network structure in GEA analyses of populations of aquatic species. We identified 146 candidate loci potentially underlying a polygenic adaptive response to seasonal fluctuations in stream flow and variation in the relative timing of temperature and precipitation extremes. Our findings underscore an emerging predominant role for seasonal variation in hydroclimatic conditions driving local adaptation and are relevant for informing proactive conservation management.     

Phylogeography of the marine isopod Stenosoma nadejda (Rezig, 1989) in North African Atlantic and western Mediterranean coasts reveals complex differentiation patterns and a new species

The transition zone between the Mediterranean and Atlantic basins has been extensively addressed in phylogeographical studies of marine species. However, biases exist towards the analysis of highly dispersive species, and there is a higher sampling effort in European coasts compared to North Africa. This may be hindering a detailed understanding of the historical and contemporary processes that shaped patterns of population genetic structure in the region. In the present study, we investigated the phylogeographical and phylogenetic patterns of mitochondrial cytochrome c oxidase subunit I sequences from a species with direct development and low dispersal abilities, Stenosoma nadejda (Rezig, 1989). The study area included 13 localities along the Atlantic and Mediterranean North African coasts, as well as the Alboran Sea. A new Stenosoma species, from the coasts of Algeria and Alboran Island, was discovered. For S. nadejda, phylogeographical analyses revealed three distinct clades: one in the Iberian Atlantic plus the Alboran Sea, one in the western Mediterranean, and another in the Atlantic coast of Africa. Haplotypes from the Alboran Island were more related to those from the western Mediterranean coast (east of the Almeria–Oran Front). Given the strong differentiation, it is probable that this species survived in multiple glacial refugia during the Pleistocenic glaciations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 419–431.